Apparatus for measuring volume of a fluid

ABSTRACT

An apparatus for transfer of a liquid using a diaphragm that separates a working fluid volume from a working air volume and can controllably induce a change pressure to draw or expel a target fluid into a tube. The apparatus is particularly suitable for automated processing of nucleic acids and other samples includes a disposable container comprising a tray and a flexible barrier. The barrier is configured to seal with a top edge of the tray, providing a closed, aseptic work area within the sealed tray. A pipette head and/or other sample manipulation device can be attached to the inside of the barrier under the diaphraghm, and the barrier can include an interface for a robotic arm or other device. When the barrier is sealed over the tray, the barrier separates the contents of the tray from the robot or other manipulation device.

RELATED APPLICATIONS

This application is a third divisional application of U.S. applicationSer. No. 12/159,748, with a 371(c) date of Feb. 12, 2009, which issuedas U.S. Pat. No. 8,030,080, on Oct. 4, 2011, which is a 35 USC 371national phase application of PCT/US2007/001170, filed Jan. 17, 2007,which claims the benefit of priority to U.S. Provisional ApplicationSer. No. 60/760,087, filed Jan. 18, 2006, through first divisionalapplication U.S. application Ser. No. 13/235,922, filed Sep. 19, 2011,and second divisional application Ser. No. 13/490,863, filed Jun. 7,2012, the contents of which are hereby incorporated by reference as ifrecited in full herein.

FIELD OF THE INVENTION

The present invention relates to automated processing of samples andmaterials, and may be particularly suitable for processing nucleic acidsin a closed environment.

BACKGROUND OF THE INVENTION

Nucleic acid based amplification reactions are widely used in researchand clinical laboratories to aid in the diagnosis of disease and/oridentification of pathogenic organisms in a test sample. Suchamplification reactions may also be used for development of vaccines,including, for example, autologous vaccines derived from a patient's owntumor cells. Amplification of nucleic acids isolated from tumor tissueallows for autologous vaccine production even from small tumors, andtherefore affords the opportunity to treat patients with minimal tumorburden.

Generally stated, the currently known amplification schemes can bebroadly grouped into two classes based on whether the enzymaticamplification reactions are driven by continuous cycling of thetemperature between the denaturation temperature, the primer annealingtemperature, and the amplicon (product of enzymatic amplification ofnucleic acid) synthesis temperature, or whether the temperature is keptconstant throughout the enzymatic amplification process (isothermalamplification). Typical cycling nucleic acid amplification technologies(thermal cycling) are polymerase chain reaction (PCR), and ligase chainreaction (LCR). Specific protocols for such reactions are discussed in,for example, Short Protocols in Molecular Biology, 2^(nd) Edition, ACompendium of Methods from Current Protocols in Molecular Biology, (Eds.Ausubel et al., John Wiley & Sons, New York, 1992) chapter 15.Isothermal reactions include transcription-mediated amplification (TMA),nucleic acid sequence-based amplification (NASBA), and stranddisplacement amplification (SDA).

Nucleic acid amplification is discussed in, for example, U.S. Pat. Nos.4,683,195; 4,683,202; 5,130,238; 4,876,187; 5,030,557; 5,399,491;5,409,818; 5,485,184; 5,409,818; 5,554,517; 5,437,990 and 5,554,516. Itis well-known that methods such as those described in these patentspermit the amplification and detection of nucleic acids withoutrequiring cloning, and are responsible for sensitive assays for nucleicacid sequences. However, it is equally well recognized that, along withthe sensitivity of detection possible with nucleic acid amplification,the risk of contamination by minute amounts of unwanted exogenousnucleic acid sequences is extremely great. The utility of amplificationreactions may be enhanced by methods to control the introduction ofunwanted exogenous nucleic acids and other contaminants.

In particular, for processing of biological samples, including forexample the production of therapeutic agents, like vaccines forautologous therapy, current good manufacturing practice (GMP) typicallyrequires manufacture in an aseptic environment.

Accordingly, there remains a need in the art to provide automatedsystems and methods for processing nucleic acids and other samples.

SUMMARY OF EMBODIMENTS OF THE INVENTION

Embodiments of the present invention are directed to systems, apparatusand methods for automated processing of one or more samples. The systemscan be used to manipulate items in a closed environment, and may beparticularly useful in the fields of medicine, diagnostics,biotechnology, electronics and nanotechnology. Embodiments of theinvention may be particularly relevant for processing biologicalsamples, including, but not limited to, tissues, blood, blood products,nucleic acids (e.g., RNA, DNA), proteins, cell cultures, and the like.

Embodiments of the present invention provide an apparatus formanipulating one or more items in a closed container. The container,also referred to herein as an “isolation container”, comprises a traydefining an interior chamber, which is configured to hold any number ofitems to be manipulated, and a flexible barrier configured anddimensioned to cover the interior area and seal with the container tray.A tool for manipulating items within the interior area is attached to orintegrated with or within the flexible barrier.

The tool can have a first interface that is accessible from an exteriorside of the barrier that is configured to attach to a robotic device.The tool can also include a second interface that can extend from anopposite side of the barrier, such that it is disposed within theinterior area when the flexible barrier is sealed with the container.The tool can be configured to manipulate items within the container whenthe robotic device is attached to the first interface. In someembodiments, the one or more items in the closed container comprise, forexample, any of nucleic acids, other samples, reagents, wash fluids,pipette tips, vessels, other consumables and/or any combination thereof.Other tools or devices may also be disposed within the container, forexample tools or devices for processing, manipulating, measuring,analyzing, sampling and/or storing samples or other items within thecontainer.

In some embodiments, systems and methods for automated processing ofnucleic acids and other samples include a single-use disposableisolation container assembly with a tray and a flexible barrierconfigured to seal with the tray, thereby providing a closed work areawithin the sealed tray. The closed work area may be aseptic.

A pipette head and/or other sample manipulation device can be attachedto the inside of the barrier, and the barrier can include an interfacefor a robotic arm or other device that is used to manipulate itemswithin the sealed work area. When the barrier is sealed over the tray,the barrier separates the contents of the tray from the robot or othermanipulation device. The barrier is flexible, and allows the robotic armto move the pipette head or other sample manipulation devices throughoutthe work area of the tray. All samples, reagents, pipette tips and otherconsumables, tools or devices for processing nucleic acid samples mayremain within the closed compartment provided by the isolation containerduring processing.

In another embodiment, methods of processing nucleic acids, (e.g., RNAand/or DNA) utilize a disposable (single-use) isolation container toreduce the risk of contaminating subject material with undesiredbiological matter, e.g., from an operator, another subject or theexternal environment. In some embodiments, the isolation container isdesigned for RNA isolation from a biological sample, including but notlimited to one or more of the following: tumor tissue, blood, bloodproducts, cells, pathogens, etc. In particular embodiments, thebiological sample comprises a tumor homogenate, and the system providesall the features and functionality to convert clarified tumor homogenateinto in vitro transcribed (IVT) RNA.

Typically, only the inside of the isolation container is exposed to thesubject material, thereby preventing possible contamination of theprocessing system and reducing cleaning requirements between consecutivesubject samples processed by the system. In some embodiments, theprocessing system processes samples in one isolation container at atime. In other embodiments, the systems can be configured to processsamples in two or more isolation containers substantially concurrently.

In some embodiments, the present invention provides an apparatus for thetransfer of fluid comprising working fluid and a working fluid pump thatare separated from a sample device by a diaphragm. In use, a sample orother fluid may be drawn into or expelled from the sample device by achange in pressure which is transmitted across the diaphragm, e.g., bymovement of the diaphragm when the working fluid pump changes pressureof the working fluid. In one embodiment, the sample device is a pipettetip or other tube for uptaking, dispensing and/or mixing fluidicsamples, and/or for transferring a sample, reagent or other fluid fromone location to another location. The pipette tip or tube may be of anysuitable shape and size.

In yet other embodiments, the present invention provides an apparatusfor measuring the volume of a fluid comprising: at least one lightsource or emitter and at least one receiver; a cuvette configured with alight path through which the receiver can detect a change in the lightpath associated with whether the cuvette contains fluid or is empty; anda fluid transfer device in communication with the receiver to determinethe volume of fluid that has been removed from the cuvette.

Other embodiments are directed to biological sample processingcontainers. The containers include a single-use disposable tray having asubstantially rigid body with a first workstation configured to hold avessel for incubation in a thermal block (e.g., a single tube,multi-well plate or strips, a PCR plate, etc.), a second workstationconfigured to hold reagents, and a third workstation configured to holdpipettes.

Some embodiments are directed to flexible barriers having an outer edgeportion configured to seal to a tray to define a sealed closed interiorchamber. The flexible barrier includes an elastomer and is sealablyattached to a robotic arm interface at a medial portion of the barrier.

Yet other embodiments are directed to an automated pipette tipdisengagement system. The system includes: (a) a tray having a sidewall;and (b) a robotic arm merging into a manipulation tool having anoutwardly extending lever configured to contact the tray sidewall,whereby contact with the sidewall forces the lever to pivot and releasea respective used pipette tip held by the manipulation tool. In someembodiments, the tray has an angled sidewall that contacts themanipulation tool lever.

Some embodiments are directed to systems for processing liquids. Thesystems include: (a) a robotic arm; and (b) a manipulation tool thatcooperates with the robotic arm, the tool configured to releasablyengage a pipette tip and automatically translate to pierce a cover on avessel a plurality of times in different spaced apart locations beforewithdrawing fluid from the vessel through one of the pierced openings inthe cover.

Other embodiments are directed to elution trays. The trays are sterilebiocompatible elution trays having a plurality of spaced apartreceptacles, a plurality on a first side of an upwardly extendingbarrier and a plurality on an opposing side of the barrier. Thereceptacles have a channel that extends on each side of and tapers downin the direction of a primary tubular portion.

Still other embodiments are directed to kits for use with an automatedprocessing system. The kits include: (a) a single-use disposablecontainer comprising a tray and a flexible barrier configured tosealably attach thereto; (b) a single-use disposable reagent rackconfigured to reside in the container at a first workstation; (c) asingle-use disposable binding column manifold configured to reside inthe container; and (d) a single-use disposable pipette rack configuredto reside in the container.

Some embodiments are directed to methods of transferring liquids. Themethods include: (a) programmatically directing a robotic arm to move aninterface tool releasably holding a pipette tip; (b) automaticallypiercing a sealant on a vessel holding a target liquid a plurality oftimes using the pipette tip; then (c) automatically withdrawing liquidfrom the vessel with the pierced sealant using the pipette tip.

Some embodiments are directed to methods of releasing liquids frompipettes. The methods include: (a) programmatically directing a roboticarm to move an interface tool releasably holding a pipette to orient thepipette in a downwardly extending angled orientation with the tipproximate to a receiving surface in a closed container; (b) thenautomatically moving the downwardly oriented angled pipette in asubstantially straight line along a plane, while releasing a flowablesubstance from the pipette.

Yet other embodiments are directed to methods of aspirating liquids intopipettes. The methods include: (a) programmatically directing a roboticarm to move an interface tool releasably holding a pipette to engage avessel holding a target fluid in a closed container; then (b)automatically moving the pipette inside the vessel to mix the liquid inthe vessel; then (c) aspirating the mixed liquid into the pipette.

In some embodiments, the method can include aspirating and dispensingthe liquid to mix the liquid (once or multiple times).

Still other embodiments are directed to automated methods of processinga sample in a closed system. The methods include: (a) providing a samplein a sealably closed container having a flexible barrier; and (b)programmatically directing a robotic arm to cooperate with the flexiblebarrier to move an interface tool inside the closed container through aseries of operations while the closed container remains sealed toprocess the sample.

The method may optionally also include one or more of the following: (c)electronically and automatically measuring volume and concentration ofthe sample at a plurality of times during the amplification; (d)electronically and automatically monitoring seal integrity of the closedcontainer before, after, and/or during use; and (e) capturing at leastone amplified RNA sample in an aliquot vessel without disrupting thesealed status of the closed system.

Still other embodiments of the invention are directed to apparatus formanipulating items in a closed container. The apparatus include: (a) acontainer having an interior region configured to hold a plurality ofitems to be manipulated; and (b) a recirculating vacuum systemconfigured to circulate air sealed within the interior region of thecontainer in a closed loop.

Although described in some embodiments herein with, respect to methodaspects of the present invention, it will be understood that the presentinvention may also be embodied as systems and computer program products.Also, it is noted that any of the features claimed with respect to onetype of claim, such as a system, apparatus, method or computer program,may be claimed or carried out as any of the other types of claimedoperations or features.

Other systems, methods, system components and/or computer programproducts according to embodiments of the invention will be or becomeapparent to one with skill in the art upon review of the followingdrawings and detailed description. It is intended that all suchadditional systems, methods, and/or computer program products beincluded within this description, be within the scope of the presentinvention, and be protected by the accompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Other features of the present invention will be more readily understoodfrom the following detailed description of exemplary embodiments thereofwhen read in conjunction with the accompanying drawings, wherein likereferences numerals represent like elements. The drawings are merelyexemplary to illustrate certain features that may be used singularly orin combination with other features and the present invention should notbe limited to the embodiments shown. Features shown with respect to oneembodiment or figure may be used with other embodiments or figures.

FIG. 1 is a perspective view of a processing system according toembodiments of the present invention;

FIG. 2 is a side view of the upper portion of the system of FIG. 1,including an isolation container on a work surface;

FIG. 3 is a perspective view of one embodiment of the work surface ofthe system of FIG. 1, illustrated without the isolation container;

FIG. 4 is a perspective view of an isolation container assembly with theflexible barrier not yet attached according to embodiments of thepresent invention;

FIG. 5 is a cutaway perspective illustration of an isolation containerassembly according to embodiments of the present invention;

FIG. 6 is a cutaway perspective view of the isolation container assemblyof FIG. 5;

FIGS. 7A and 7B are side views of a syringe pump system according toembodiments of the present invention;

FIGS. 8A and 8B are cross-sectional side views of a pipette head adaptedto cooperate with a fluid pump system according to embodiments of thepresent invention;

FIG. 8C is a top view of a flexible isolation diaphragm used in thepipette head of FIG. 8A, according to embodiments of the presentinvention;

FIG. 8D is a cross-sectional view of the flexible isolation diaphragmshown in FIG. 8C;

FIGS. 9A and 9B are perspective and cross-sectional views, respectively,of a flexible barrier according to embodiments of the present invention;

FIGS. 9C and 9D are perspective and cross-sectional side views,respectively, of other embodiments of a flexible barrier;

FIG. 9E is a perspective view of a flexible barrier with an integralcoupler, the coupler in the barrier shown in partial section view,according to embodiments of the invention;

FIG. 9F is a perspective view of a portion of a robotic arm having acoupler configured to engage the barrier coupler shown in FIG. 9Eaccording to embodiments of the present invention;

FIG. 9G is a perspective view of the assembly of the components shown inFIGS. 9E and 9F (without the flexible barrier) and with the outerhousing over the internal components shown transparent and in brokenline;

FIG. 9H is a perspective view of the assembly shown in FIG. 9G with theflexible barrier attached according to embodiments of the presentinvention;

FIG. 10 is a cross-sectional view of a PCR plate and thermal cycler lidassembly according to embodiments of the present invention;

FIG. 11 is a perspective view of a thermal cycler assembly with athermal cycler lid drive system according to embodiments of the presentinvention;

FIG. 12 is a perspective view of the thermal cycler lid drive system ofFIG. 11 in use with a flexible thermal cycler lid seal of a containeraccording to embodiments of the present invention;

FIGS. 13A and 13B are cross-sectional views of the lid seal of FIG. 12during movement of the thermal lid into and out of the work space of thecontainer according to embodiments of the present invention;

FIG. 13C is a partial cutaway side perspective view of the lid seal andcontainer shown in FIGS. 13A and 13B.

FIG. 14 is a perspective view of a manifold for housing DNA and RNAbinding columns according to embodiments of the present invention;

FIG. 15 is a perspective view of the manifold of FIG. 14 duringengagement with a pipette head to move the manifold between stationsaccording to embodiments of the present invention;

FIG. 16 is a schematic side view depicting a closed vacuum system priorto use with an isolation container according to embodiments of thepresent invention;

FIG. 17 is a schematic side view of the vacuum system of FIG. 16 duringuse with the isolation container according to embodiments of the presentinvention;

FIG. 18 is a perspective view of a processing system work surface with acutaway of an isolation tray, showing cooling units for controllinghumidity and vapor concentrations according to embodiments of thepresent invention;

FIG. 19A is a cross-sectional perspective view of a cuvette attached toan isolation tray according to embodiments of the present invention;

FIG. 19B is a side perspective view of cooperating components of aspectrophotometer cuvette measuring system according to embodiments ofthe present invention.

FIGS. 20A-20D are examples of operations of a system to measure volumein a volumetric cuvette according to embodiments of the presentinvention, each figure being a section view of the volumetric cuvettemeasuring system;

FIG. 20E is a top, side perspective view of a multi-cuvette volumemeasurement system that can operate as described with respect to FIGS.20A-20D according to embodiments of the invention;

FIG. 20F is a bottom perspective view of the container shown in FIGS. 4and 5 illustrating cuvettes extending below the bounds of the containeraccording to embodiments of the present invention;

FIG. 21A is a perspective view of a reagent rack according toembodiments of the present invention;

FIG. 21B is a schematic top perspective view of the reagent rack shownin FIG. 21A illustrating a piercing technique according to embodimentsof the present invention.

FIG. 22 is a series of schematic illustrations showing operation of analiquot tube mechanism according to embodiments of the presentinvention;

FIG. 23A is an illustration showing components that may be loaded intoan isolation container tray according to embodiments of the presentinvention;

FIG. 23B is a top perspective view of a partially assembled containerusing the kit components shown in FIG. 23A according to embodiments ofthe present invention;

FIG. 23C is a bottom view of the partially assembled kit shown in FIG.23B;

FIG. 23D is a top view of the container in the kit shown in FIG. 23Abefore kit components are attached at a use site according toembodiments of the invention;

FIG. 24 is an exploded view of the isolation container tray of FIG. 23Aillustrating sealing of the tray with a flexible barrier according toembodiments of the present invention;

FIG. 25 is a flow chart of operations that can be carried out accordingto embodiments of the present invention;

FIGS. 26A and 26B are enlarged side perspective views of the pipettehead with lever according to embodiments of the present invention;

FIG. 26C is a side perspective view of the lever shown in FIGS. 26A and26B shown engaging with an interior surface of the container to releasea pipette tip according to embodiments of the present invention;

FIG. 27 is a top perspective view of a trolley that can be used to loadthe container onto an instrument according to embodiments of the presentinvention;

FIG. 28A is an enlarged side perspective view of a work surface with abiasing assembly configuration that can force the container intoalignment with the robotic arm according to embodiments of the presentinvention;

FIG. 28B is a bottom perspective view of a portion of the work surfaceshown in FIG. 28A illustrating a spring used to force the container in adesired direction according to embodiments of the present invention;

FIG. 29A is a top view of an elution tray according to embodiments ofthe present invention;

FIG. 29B is a bottom perspective view of the tray shown in FIG. 29A;

FIG. 29C is a top perspective view of the tray shown in FIG. 29A;

FIG. 30A is a top perspective view of a waste tray cover;

FIG. 30B is a bottom perspective view of the tray cover shown in FIG.30A;

FIG. 31 is a block diagram of a circuit used in automated systemscontemplated by the present invention; and

FIG. 32 is a block diagram of a data processing system according toembodiments of the present invention.

DETAILED DESCRIPTION

While the invention may be made in modified and alternative forms,specific embodiments thereof are shown by way of example in the drawingsand will be described in detail. It should be understood, however, thatthere is no intent to limit the invention to the particular formsdisclosed, but on the contrary, the invention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the invention. Like reference numbers signify like elementsthroughout the description of the figures.

In the figures, the thickness of certain lines, layers, components,elements or features may be exaggerated for clarity. Broken linesillustrate optional features or operations or hidden components unlessspecified otherwise. In the claims, the claimed methods are not limitedto the order of any steps recited unless so stated thereat.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof. As used herein, the term “and/or”includes any and all combinations of one or more of the associatedlisted items. As used herein, phrases such as “between X and Y” and“between about X and Y” should be interpreted to include X and Y. Asused herein, phrases such as “between about X and Y” mean “between aboutX and about Y.” As used herein, phrases such as “from about X to Y” mean“from about X to about Y.”

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the specification andrelevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. Well-known functions orconstructions may not be described in detail for brevity and/or clarity.

It will be understood that, although the terms first, second, etc. maybe used herein to describe various elements, components, regions, layersand/or sections, these elements, components, regions, layers and/orsections should not be limited by these terms. These terms are only usedto distinguish one element, component, region, layer or section fromanother region, layer or section. Thus, a first element, component,region, layer or section discussed below could be termed a secondelement, component, region, layer or section without departing from theteachings of the present invention. The sequence of operations (orsteps) is not limited to the order presented in the claims or figuresunless specifically indicated otherwise.

The terms “closed system” or “closed container” refers to systems andcontainers, respectively, that are sealed and operate in asubstantially, if not totally, closed manner to inhibit or prevent theintroduction of exogenous or external materials into (or out of) thesystem or container during processing. The closed systems can beconfigured to inhibit or prevent contamination. In some embodiments,components of the closed systems or containers can be pre-sterilizedprior to use at a manufacture site, sterilized at the point of use,and/or sterilized after a respective closed system is assembled andclosed prior to use. The closed systems or containers can be pressure-or vacuum-tight to some target or predefined leak rate at certainatmospheric conditions. The leak rate may be detected by an internaland/or external sensor using a vacuum or pressure sensor test or otherleak check system and the like. The typical atmospheric conditions maychange with location of the apparatus, from sea level to higheraltitudes. However, the closed systems contemplated by embodiments ofthe invention may be used in marine (subsurface, deep sea), flight,and/or space environments as well, with the seal being sufficient tosubstantially maintain the target leak rate at those atmosphericconditions. The closed systems or containers can be utilized for theirrespective intended purpose without breach to the integrity of theclosed system. The closed systems or containers may be adapted for fluidtransfers of target fluid samples in or out while maintaining asepsis,and/or can be connectable to other closed systems while maintaining theintegrity of the closed systems. Filters may optionally be used in aflow path that may be open to atmosphere or other components duringprocess. The filters may be configured to filter to a desired cleanlevel or class, to facilitate the closed state, such as class 100,000,class 10,000, class 1000 filters or even class 100 filters.

The term “isolation container” refers to a container configured to hold,enclose and/or isolate internal components and the processing of one ormore items or samples held therein from external pathogens,microorganisms and the like and/or other materials that may exist in anexternal environment. The isolation container may be a single-usedisposable container. In some embodiments, the samples in the isolationcontainer are or comprise nucleic acids, e.g., for processing nucleicacids in tissues (e.g., clarified tumor homogenate) into IVT RNA.

The term “single-use disposable” refers to a component that is notreused. That is, after completing its intended use, i.e., processing orproduction of a target sample or sample(s), it is disposed of. Theisolation container can be single-use disposable (and may be labeled assuch), such that the isolation container remains a closed system that isdisposed of in the closed sealed state with its internal components heldtherein, to inhibit any inadvertent external release or exposure of itsinternal content(s) after processing/production of the target product.

The term “aliquot” refers to a desired amount of a target fluid; theamount of fluid may be in a predetermined and/or specific range. Theterm “aliquot tube” refers to a tube that permits removal of a fluidaliquot from the container. Typically, the term “aliquot tube” refers toa tube that is in communication with the interior of the isolationcontainer to receive at least one (and typically a plurality of) fluidaliquot without breaching the closed status or sealed integrity of theisolation container. The aliquot tube can be flexible and sterile andmay comprise an elastomer, such as PVC.

The term “cuvette” refers to a vessel configured to permit mechanical,electrical or optical measurement(s) of a substance, typically a fluid,and typically concentration or volume measurements, contained withinthat vessel. The cuvette may be sized to hold a relatively small amountof fluid, typically, in the range between about 0.001 mL to about 5.0mL.

The term “pipette tip” refers to a tube open at both ends to be able tointake and/or discharge fluid, typically liquids in small volumes, andtypically in amounts between about 0.1 μL to about 1000 μL. The tube mayhave an irregular or constant perimeter shape or size, typicallytapering down at a lower tip from the head. A “pipette” can be definedby a plurality of matable components. That is, a pipette can include atip portion and a head portion. The head portion can be defined on amanipulation tool attached to a robotic device (indirectly) thatreleasably attaches to different pipette tips during processing.Pipettes of different volumetric sizes may be used in a singlecontainer/processing system.

The term “Human Machine Interface (HMI)” is well known to those in theart and refers to an interface which allows an operator to input,direct, interact with or machines, and typically includes an electronicdisplay with a “Graphic User Interface (GUI)” that programmaticallyprovides information to and accepts control instructions from anoperator.

The term “aseptic” refers to processing conditions that inhibit orprevent contamination of a target sample in an interior processing spaceof a container by external pathogenic microorganisms and/or undesiredexogenous materials, and/or to inhibit or prevent contamination of theproximal exterior environment with the contents of the container.

The term “binding column” refers to a filtration/elution column that canbe used for separating components of a sample or derivatives thereof. Insome embodiments, the binding column can be used for nucleic acid (e.g.,DNA and/or RNA) isolation or purification. In some embodiments, thebinding column may contain a silica membrane.

The term “tray” refers to a substrate having sufficient rigidity to holdone or more components. The tray may be substantially flat or may have abowl-like shape. The tray may also have other shapes and configurations.The tray can be configured to define integral wells or holding regionsor may be configured to sealably mate with and/or hold devices orcontainers, typically those components associated with at least oneprocessing workstation.

The term “user” is a generic term for an operator, programmer, and/ormaintainer.

The term “robot” refers to an automated device that can beprogrammatically directed to translate in desired directions to carryout defined processing steps or operations. The term “robot” is usedbroadly and includes a stationary mounted robotic arm with a multi-axistranslation as well as a fully mobile robot and other appropriaterobotic devices.

The present invention may be embodied as systems, methods, and/orcomputer program products. Accordingly, the present invention may beembodied in hardware and/or in software (including firmware, residentsoftware, micro-code, etc.). Furthermore, the present invention may takethe form of a computer program product on a computer-usable orcomputer-readable storage medium having computer-usable orcomputer-readable program code embodied in the medium for use by or inconnection with an instruction execution system. In the context of thisdocument, a computer-usable or computer-readable medium may be anymedium that can contain, store, communicate, propagate, or transport theprogram for use by or in connection with the instruction executionsystem, apparatus, or device.

The computer-usable or computer-readable medium may be, for example butnot limited to, an electronic, magnetic, optical, electromagnetic,infrared, or semiconductor system, apparatus, device, or propagationmedium. More specific examples (a non-exhaustive list) of thecomputer-readable medium would include the following: an electricalconnection having one or more wires, a portable computer diskette, arandom access memory (RAM), a read-only memory (ROM), an erasableprogrammable read-only memory (EPROM or Flash memory), an optical fiber,and a portable compact disc read-only memory (CD-ROM). Note that thecomputer-usable or computer-readable medium could even be paper oranother suitable medium, upon which the program is printed, as theprogram can be electronically captured, via, for instance, opticalscanning of the paper or other medium, then compiled, interpreted, orotherwise processed in a suitable manner, if necessary, and then storedin computer (electronic) memory.

Apparatus and methods for automated processing in a closed environmentare discussed below. The systems can be used, for example, to process,fabricate, assemble or otherwise manipulate anything in a closedenvironment, and is particularly useful in the fields of medicine,forensics, therapeutics, diagnostics, biotechnology, electronics andnanotechnology. For convenience of description, various aspects andfeatures of the invention are described herein in the context of asystem for processing nucleic acids. One skilled in the art willappreciate, however, that the following descriptions are intended to bemerely illustrative of the invention and not restrictive. Various otherapplications are intended within the scope of the present invention,including, for example, systems and methods for fabricating, assembling,processing or otherwise manipulating any items in a closed container.

Overview of Exemplary Automated Systems

In overview, referring to FIG. 1, the system 10 can be configured with arobotic arm 20 that manipulates one or more items within a closedcontainer assembly 12 with an interior chamber (also described as aninterior work area or region, where the word “area” is used broadly andnot in a two-dimensional mathematical manner). In some embodiments, thecontainer assembly 12 has an interior region 18 configured to hold aplurality of items to be processed and/or otherwise manipulated. Aflexible barrier 16 can be configured and dimensioned to cover theinterior of and seal with a container tray 14. A tool for manipulatingitems within the chamber is attached to or integrated within theflexible barrier 16 and can extend into the interior region when thebarrier 16 is attached to the tray 14. The tool can have an adapter orother interface that is accessible from an exterior side of the barrier,such that the tool may be manipulated by a robotic device from outsideof the container when the barrier is sealed with or otherwise attachedto the container tray. The tray 14 may be configured to include one ormore workspaces, areas, stations, racks, holders, receptacles, wells orother devices for holding one or more items or process components orfluids in the closed container. Other tools or devices may also bedisposed or otherwise accessible from within the container; for example,tools or devices for processing, manipulating, measuring, analyzing,sampling and/or storing samples or other items may reside within thechamber of the container.

Turning now to the figures, FIG. 1 shows an exemplary automated nucleicacid processing system 10. System 10 may include a container assembly12. The container assembly 12 may be an isolation container 12. Thesystem 10 also includes a robot 20 (typically a robotic arm) or otherautomated or manually directable device for manipulating items withinand/or associated with the container assembly 12. The container assembly12 comprises the tray 14 and the flexible barrier 16. The barrier 16 isconfigured to seal with or otherwise attach to the tray 14. An outerperimeter edge portion 16 p of the barrier 16 can sealably engage thecontainer tray 14, providing a closed, aseptic work area 18 within thecontainer assembly 12. In some embodiments, the container assembly 12,or any portion thereof, is single-use disposable. In particularembodiments, the sample can be discharged, collected or captured outsideof the container without opening the sealed container, and the sealedcontainer 12 can be disposed of “intact” with the remainder internalcontents.

The tray 14 can be substantially rigid and may be a molded body. In someembodiments, the tray 14 comprises a medical grade (such as USP Class VIor other suitable grade) molded polymer. The body of the tray 14 maycomprise one or more substantially clear, translucent or transparentpolished regions for optic visibility that provides for internalviewability. The tray 14 can be molded in a clean room mold and in aclean room molding facility to inhibit and/or minimize bioburden andparticulates. The tray 14 may be sterilized prior to or after sealingthe flexible barrier 16, by using conventional sterilization techniques,such as, for example, surface decontamination with VHP (vaporoushydrogen peroxide), gamma irradiation or ethylene oxide vapor hydrogenperoxide. For shipment, the tray 14 may be enclosed in doubleelastomeric sterile packaging material, such as such as sealed doubleplastic wrapping/bagging.

The barrier 16 can be configured to allow the robot 20 to move a toolinside the work area 18 of the sealed container assembly 12 betweenabout 4-24 inches vertically, and horizontally at least about the widthand length of the tray 14, without destroying the sealed integrity ofthe container assembly 12. In some embodiments, the robot 20 can directan internal interface to have between 6-12 inches of vertical movementin the sealed container assembly 12 without destroying the sealedintegrity of the seal. The barrier 16 can be sized and configuredrelative to the tray 14 to provide for a sufficient volume of air toallow the desired range of movement of the robot 20 without overlycompressing or decompressing the interior volume of the container 12.

In some embodiments, the system 10 is used to process biologicalsamples, including, but not limited to tissues, blood, blood products,nucleic acids, proteins, cell cultures, and the like inside thecontainer assembly 12. In such embodiments, the container 12 maycomprise, for example, items such as biological samples, reagents, washfluids, pipettes, pipette tips, vessels, other consumables or anycombination thereof.

As shown, the system 10 can be a self-standing, self-contained unit. Thesystem 10 can include a housing or chassis 70 for supporting anisolation container assembly 12 and other components of the automatedsystem 10, such as, for example, the robot 20, a thermal block 30 (e.g.,a heat block and/or cooling block, etc. . . . . ), an aliquot retrievalassembly 40 and related mechanisms for collecting processed samples, auser interface 60, and various other components and mechanisms asdescribed in the sections that follow. The thermal block 30 maycooperate with a thermal block assembly 1000 as shown, for example, inFIGS. 10, 11.

In some embodiments of a processing system 10, an operator or maintainermay only need to access the front of the system 10 for normal operation.As shown in FIG. 1, one or more access doors 110 may open, e.g., bylifting substantially vertically or opening to a side or in anotherdirection, to provide accessibility while installing or removing anisolation container 12.

In some embodiments, due to the automated nature of system 10, the door110 may protect the operator from moving parts of the system duringoperation. The system 10 may incorporate an electronically controlledinterlock or other mechanism to inhibit or prevent the operator fromopening the door 110 while the system is in operation. A manual overridemay be provided to allow opening of the interlocked guards, and such anoverride may be employed, for example, if no power is available. Theuser interface 60, also referred to as an HMI (Human Machine Interface),can be configured to allow an operator to initiate a processingoperation or otherwise control the system 10, and can be mounted on thefront of the chassis 70 next to the access door 110.

In one embodiment, the display screen 61 and controls (touchscreen,keypad or other input) of the HMI may be positioned to allow the user toview and operate them while standing. In some embodiments, the rear andsides of the system 10 do not need to be routinely accessed byoperators. Consequently the system can be positioned against a wall oranother system.

In some embodiments, a lower section of the chassis 70 can containelectronic control systems, power supplies and other components, whichmay not require operator interaction. These components can be housed indedicated enclosures that protect the components from accidental fluidingress and protect the operator from possible electrical hazards.Removable panels 120, 130, 140 may be included to provide access to theelectrical components and/or other components inside the enclosures. Insome embodiments, only trained service personnel are permitted orrequired to access the insides of these enclosures. The service accesspanels may or may not be interlocked, for example, to allow or disallowthe system to continue to operate with the panels removed.

FIG. 2 shows an enlarged side view of an upper portion of system 10,including isolation container 12 on a work surface 200. A pipette head220 and/or other manipulation device can be attached to or integratedwith or within the barrier 16, for example, such that at least a portionof a pipette head 220 extends into the interior work area 18 ofcontainer 12 when the barrier is attached to the tray 14 as shown. Thepipette head 220 can include an interface on an opposite side of barrier16 that is configured to releasably connect with an adapter 210 on anend portion of arm 230 of robotic device 20. In some embodiments, apipette head 220 or other manipulation device does not physically passthrough the barrier 16, and may not necessarily couple to a robot orother device through an adapter. Rather, such a pipette head may attachto or otherwise integrate with the inside of the barrier, and may beoperated or manipulated from the opposite side of the barrier 16, forexample, using the robotic arm 230 attached to the opposite side of thebarrier. In other embodiments, a pipette head or other samplemanipulation device integrated with the barrier 16 (which can besubstantially impermeable) may be operated by a magnet or other devicethat supports and/or communicates with the manipulation device throughthe barrier 16.

When the barrier 16 is sealed over the tray 14, the barrier 16 separatesthe contents of the tray 14 from the robot 20 or other device thatmanipulates the pipette head 220. The seal between barrier 16 and tray14 can be airtight, thereby providing a closed environment within thecontainer 12. In some embodiments, the barrier 16 is flexible and allowsthe robotic arm 230 to move the pipette head 220 throughout the workarea 18 within tray 14. All items or materials involved in processingthe nucleic acid samples, including for example biological samples,reagents, pipette tips, or other consumables, and other tools or devicesfor processing the samples, can remain within the closed work area 18during processing. The tray 14 may include a number of features orstations 242 which may be molded, formed, attached or otherwiseintegrated with base 240 of the tray 14, or another portion of the tray14, to accommodate such items.

As shown, robotic device 20 may be attached to a wall of the chassis 70.In other embodiments, robotic device 20 may comprise an arm, gantry orlinkage system attached to work surface 200 or to another supportstructure. Suitable robotic devices are known in the art. Non-limitingexamples include Stdubli TX90 robot, Epson E2L Scara robot, Kawasaki FSeries robots, Yamaha YKL Scara robot, ST Robotics R17 Robot, etc. Insome embodiments, the robotic device 20 is a FANUC LR MATE 200iB 5C,6-axis robot or other suitable multi-axis robotic device, and is used toautomatically perform the desired manipulations inside the closedcontainer 12, such as, for example, aspiration and transfer of fluidsusing pipette tips and sliding or lifting a binding column manifoldduring processing of samples.

The multi-axis robotic arm can be used to move an interface toolreleasably holding a pipette to orient the pipette in a number ofdifferent directions to help mix the liquid upon aspiration ordispensing. For example, the pipette can be oriented in a downwardlyextending angled orientation with the tip proximate to a receivingsurface in a receiving vessel in the closed container, then,automatically, the downwardly oriented angled pipette can be moved in asubstantially straight line along a plane (similar to a typicallylayering or dispensing of mustard), while releasing a flowable substancefrom the pipette. In other embodiments, the robotic arm canautomatically move the pipette to mix the liquid in the vessel prior toaspiration, then aspirate the mixed liquid into the pipette.Alternatively or additionally, in some embodiments, the liquid can beaspirated and dispensed at least once to mix the liquid. In someembodiments, the receiving or dispense vessel has a lip, and the roboticarm is configured to direct the pipette tip to move around the perimeterof the lip.

As shown in FIG. 3, the horizontal work surface 200, which may belocated inside of front access door 110, for example, supports variouscomponents and mechanisms that interact with an isolation container 12.The work surface 200 can include one or more guides 390, that mayinclude guide slots 390 s that engage components on an assembly supporttrolley 399 (FIG. 27) to align the container, or other location featuresto orient and releasably clamp to the isolation container 12 (e.g., tobase 240 of FIG. 2) during processing.

As shown in FIG. 27, a container assembly 12 can be prepared on atrolley 399 that can dock with the system 10. The trolley 399 caninclude slots 399 s that align with slots 390 s defined by guide 390 ona support surface of the system 10. To transfer the container assembly12 to the system 10, an operator can roll the trolley adjacent to thesystem 10, so that the trolley 399 is aligned with the guide 390 of thesystem 10. The container assembly 12 can slide into the slots 390 s ofthe system 10 into proper operative position without requiring that anoperator lift the container assembly 12.

As shown in FIGS. 28A and 28B, the work surface 200 can include rollerguides 381, 382 that contact the lower portion of the container 12 whenit is in position. The roller guide 382 can be in communication with aspring 383 that can bias the container 12 inwardly so as to fit snuglyagainst the innermost slot wall 390 to be in the desired alignment withthe robotic arm for registration of workstation locations.

Referring again to FIG. 3, the work surface 200 may support atemperature control device, such as an incubation device for heatingand/or cooling regions, vessels, or target objects or locations withinthe container. Such devices are known to those of skill in the art. Inone embodiment, the system 10 includes a thermal cycler 30 forcontrolling temperature of a PCR plate 610 (FIG. 6) or other vessel orcomponent during some nucleic acid amplification reactions or otherreactions or processes, for example, and a thermal cycler lid mechanism310 for selectively covering the thermal cycler 30 during suchreactions. In one embodiment, a vacuum pump assembly 320 and a pinchvalve assembly 330 provide for control of vacuum and circulation of airwithin the isolation container 12 during processing. The vacuum pumpassembly 320 can releasably engage a single-use disposable vacuum head1602 and associated tubing 1650 (FIG. 23B). The work surface 200 canalso hold the container assembly 12 so as to allow a spectrophotometer1900 s to communicate with components held by the container assembly 12as will be discussed further herein.

One or more syringe pumps 350 (shown, for example, as two pumps) may beused to drive aspiration and dispense actions of pipette head 220 (FIG.2). In some embodiments, syringe pumps 350 may be used to pump a workingfluid, e.g., from a reservoir 340, through the pipette head adapter 210shown in FIG. 2 to provide vacuum and pressure for operation of pipettehead 220. The working fluid may be substantially incompressible, andcan, for example, comprise an aqueous solution of about 50% ethanol.Other fluids, even air, may be used. Additional details of an exemplaryembodiment of a pump mechanism and pipette head 220 are described ingreater detail below in connection with FIGS. 7 and 8.

Returning to FIG. 3, a cuvette holder 360 (see also spectrophotometercuvette in FIG. 19A) can be coupled with a spectrophotometer 1900 stypically residing in a lower portion of the chassis 70, to provideautomatic concentration measurements at desired operations duringprocessing. Optionally, the work surface 200 may include one or morecooling features, e.g., a heat exchanger such as a chiller or Peltiercooling plates 370 and 372 are positioned to engage certain portions ofthe isolation container 12 and help control humidity and vaporconcentrations within the sealed container 12. A lever 380 or othermechanism or device may be used to secure the isolation container intoplace on the work surface 200.

In some embodiments, an RNA processing system may operate in astand-alone fashion isolated from a larger production system and anydata management tools or systems. An internal or external computersystem may control and/or monitor the automated components of thesubsystem. Users may interact with the interface 60, for example, tooperate the system and monitor status. In other embodiments, the system10 can communicate with other systems or a monitoring station (which maybe in a different room or even in a different facility).

Additional details of the various components and assemblies, andexemplary methods of use, are described in sections that follow.

Exemplary Isolation Containers

Referring to the isolation container assembly 12, tray 14 and flexiblebarrier 16 are described in greater detail. The tray 14 includes aninterior work area 18 and may comprise a number of inserts, recesses,racks, or rack mounting features and/or stations 242 for holding itemswithin container 12, e.g., including one or more racks, manifolds orother holders for holding vessels, pipette tips, binding columns, othersubstrates and/or other consumables or devices for a desired assay orprocess. All items used to process the material to a desired finishedstate or product can be held within the container, i.e., the consumablesfor the system can be self-contained and sealed during processing tominimize the risk of contaminating the interior of the container, or toprevent a sample from contaminating the environment or user. The barrier16 can have an outermost perimeter portion 16 p (FIG. 5) that can beconfigured to releasably or permanently seal with the tray 14, forexample about an annular edge 452 of tray 14 as shown in FIG. 5. Whenthe barrier 16 is sealed with the tray 14, at least a portion of pipettehead 220 and/or another sample manipulation tool or device extends fromthe barrier 16 into the work area 18.

In some embodiments, the barrier 16 engages the tray 14 along a rigidtop edge 452 (FIG. 5). The outermost perimeter portion 16 p of thebarrier 16 can reside against the top edge 452 (FIG. 5). The top edge452 may be configured as an upstanding lip with a gap space thatreceives an O-ring (not shown) that pinches against the barrier 16 toseal the barrier against the tray 14.

To create a closed system or environment, the isolation container tray14 and barrier 16 may be configured to form a physical barrier betweenitems within the container 12 and external devices, mechanisms andpieces of equipment used for processing the items in the container. Forexample, a flexible thermal cycler lid seal 430 may be integrated withina wall 450 of the tray 14 as shown in FIG. 4. Such a seal 430 mayenclose and define a sleeve or closed channel that receives the lid 1010(FIG. 11) and be used to allow the thermal cycler lid mechanism 310 toextend the lid into an aperture 14 a (FIG. 5) in the sidewall of thetray 14 to cover samples during heating by a thermal cycler block 30,while providing a physical barrier between the lid mechanism 310 anditems within the interior area 18 of container 12.

Also, cooling plates 370, 372 may engage portions of an external surfaceof the tray 14 to impart changes in temperature within the container 12while not contacting any items within the isolated work area. In someembodiments, cooling plates may engage with portions of the externalsurface of the tray 14 and/or container 12 to control the temperature ofitems (such as reagents) held inside the isolated work area/space.

Similarly, as shown in FIG. 3, a cuvette holder 360 can selectivelyengage a cuvette 660 (FIG. 6) extending from base 240 of tray 14 toperform measurements of concentration and/or volume, but the holder 360may not contact any items within container 12. Thus, in someembodiments, only the reagents, consumables and tools or devices thatare required to directly contact the sample are contained within theisolation container. Such a configuration may have several benefits. Forexample, a sample can be processed in its own isolated environmentand/or the processing equipment residing outside the container will notcontact the sample and so is protected from possible contamination. Atthe conclusion of processing, a desired number of aliquots may becollected in aliquot tubes 440 (FIG. 4) and removed from the container12, e.g., without compromising the integrity of the collected samples orthe isolation container, as will be discussed further below. In someembodiments, for example, the sealed isolation container 12, along withthe components contaminated by sample material, can be discarded after aprocessing cycle.

FIGS. 5 and 6 show additional details of some components of exemplaryisolation containers 12, including the isolation tray 14 and theflexible barrier 16. The interior work area 18 may be surrounded, forexample, by base 240 and one or more walls 450. The barrier 16 caninclude a pipette head 220 or other device or tool, which is describedin more detail in a separate section below. The barrier 16 can beconfigured to cover and seal with the tray 14, for example at a seal 510along an annular edge 452 of the tray 14, such that the work area 18 isa closed, typically air-tight, compartment when the barrier 16 is sealedwith the tray 14.

The tray 14 can be substantially rigid and includes a number of featuresand/or stations 242 which may be molded, formed, attached or otherwiseintegrated with tray to accommodate. Some or all of the followingcomponents are examples of features that may be integrated into the tray14:

-   -   elution 630 and waste 632 stations;    -   vacuum inlets and outlets;    -   spectrophotometer (concentration) cuvettes 660 (FIG. 19A);    -   other vessels or cuvettes, e.g., volume measurement cuvettes        2060 (FIGS. 20A-E);    -   a thermal cycler PCR plate 610 and/or other substrate or vessel        that may be heated and/or cooled with a temperature control        device;    -   reagent vessels 622 in a rack 620;    -   pipette tips 651 in a rack 650;    -   one or more additional tip racks 650 or other disposal        container, e.g., for used pipette tips 653;    -   a binding column manifold 50, e.g., which may include one or        more binding columns for nucleic acid processing; and    -   one or more aliquot tubes 440 for processing outputs: e.g.,        nucleic acids, such as (e.g., DNA, RNA, tumor total RNA and/or        amplified tumor in vitro transcribed RNA and the like),        microorganisms, cells, medicaments, etc.

In some embodiments, the flexible barrier 16 seals with and supports amanipulation tool that interfaces with the robotic device 20 on one sideof the barrier and interfaces with internal components on the inside ofthe barrier 16. The tool can include the pipette head 220 and additionalmanipulation features, such as handles 520 or other features ormechanism for engaging internal components, such as, for example,handles 50 h (FIG. 5) on a binding column manifold 50 for transferringthe manifold between the waste and elution stations 632, 630,respectively. The flexible barrier 16 can allow the pipette head 220 tomove sufficiently to access all desired internal components housed bythe tray 14. The head 220 can also engage the binding column manifold 50to be able to transfer the binding column manifold to and/or from thewaste 632 and elution 630 stations. Although not shown, the internalinterface may be a plurality of serially attachable interfaces. That is,the pipette head or first interface can be releasably held andinterchanged for another manipulation tool inside the closed container.The selection of manipulation tools can be held by tray 14 and/or aninternal manipulation tool rack (not shown).

A mixer and/or centrifuge may cooperate with or be integrated into thetray to mix, homogenize, separate or otherwise blend or process a liquidor sample, such, as for example, a biological sample or other materialsas desired (not shown). In other embodiments, the starting material maybe pre-mixed, and then placed in the rack 620 or at another location.The mixer may be a rotating head mixer, a magnetic mixer or ahomogenizer that can mix the desired material.

Exemplary Pipetting Systems

Referring to FIGS. 7A and B, a pipetting system 700 comprising a pipettehead 220 and adapter 210 assembly may be used to perform fluid transfersinside the isolation container 12. In keeping with a closed isolationcontainer design, the pipette head 220 can be configured to maintain abarrier between the fluid being transferred and the pipette pumpmechanism. A filter and/or a flexible diaphragm 710 can provide thephysical contamination-resistant barrier between the interior of theisolation container 12 and the outside.

In some embodiments, to provide high volumetric accuracy, the pipettingsystem 700 may use two positive displacement syringe pumps 350-1, 350-2,e.g., one for low volume transfers (e.g., between about 1 μl and about50 μl) and one for the higher volumes (e.g., between about 51 μl andabout 1000 μl). In other embodiments, a single pump chamber withdifferent reservoirs or a single reservoir with a means of metering theworking fluid can be employed. The pump chambers may be hydraulicallyconnected to the flexible diaphragm 710 via (substantially rigid) tubingand a buffer solution (working fluid). The buffer solution can be asubstantially or totally incompressible hydraulic fluid that can reduceor minimize the elasticity of the pipetting system 700. The robotic arm230 is used to position the pipette head 220 at desired locations in theworkspace. To reduce the potential for reagent carryover during pipettetransfers, disposable tips 651 may be used for fluid handling. That is,after each transfer, the used tip can be discharged into a “trash”receptacle, typically into a used rack, and a new sterile different tipfrom a sterile rack or supply station may be used for the next transfer.Other components of an exemplary embodiment of the pipetting system 700are shown in FIGS. 7A and 7B.

In some embodiments, the pipetting system 700 is primed to introduceliquid and remove air from the pipette line prior to initial use, andmay be primed prior to each time a different pipette head 220 isconnected to the pipette head adapter 210, such as at the beginning ofprocessing of a new closed container 12. For example, a bleed lineconnects a bleed port in the pipette head to the working fluid reservoirvia a solenoid valve 741. The valve 741 may be opened during the primingsequence to allow air to be bled from the pumps 350, through the fluidlines 222, 223 (FIG. 8A) and pipette head 220, and back to the workingfluid reservoir 340. The robotic arm 230 can be directed to orient thefluid lines in the pipette head 220 so that they are angled during thepriming. Once the system 700 is primed, the bleed valve 741 may beclosed and the pipette head 220 is ready to perform fluid transfers. Atthe completion of the RNA process, the bleed valve 741 may be used todrain the fluid lines 222, 223 and pipette head 220. Filling the fluidlines with air may prevent the possibility of fluid spills duringdisconnection of the pipette head.

FIGS. 8A and 8B show a cross-sectional view of the operation of apressure transfer mechanism 800 for transferring pressure through thepipette head 220 and to the pipette tips 651, 653. In some embodiments,the hydraulic working fluid, e.g., from reservoir 340, actuates theflexible isolation diaphragm 710 (see also FIGS. 8C and 8D) and drivesthe pipetting action while maintaining physical separation between theworking fluid and the fluids to be aspirated by the pipette. A reservoirof working fluid can reside on one side of the diaphragm 710, which issealed from working air on the other side. The flexible diaphragm 710resides in the head 220 between the working fluid 340 and the internalpipette tip adapter 220 a (i.e., pipette head) and aspirated fluid. FIG.15 illustrates the adapter 220 a without the diaphragm and upperassembly.

Referring again to FIGS. 8A and 8B, displacement of the diaphragm 710affects the working air volume 700 a under the diaphragm 710 and in thepipette tip adapter 220, thereby causing the pipette tip to intake ordischarge (typically meted) amounts of liquid. The overall elasticity ofthe pipetting system 700 may be directly related to dispense accuracy.In some embodiments, reducing the air volume 700 a can improve both thepipetting accuracy and precision.

In some embodiments, to facilitate thorough mixing of fluids withinreagent vessels, spectrophotometer cuvettes 660 and PCR wells, arepeated aspirate/dispense cycle can be employed and/or the robotic arm230 can be directed to move the tip of the pipette in multi-axistranslation.

In some embodiments, the tray 14 and/or pipette head 220 may include oneor more features to allow used tips 653 to be removed from the pipettetip adapter 220 a (FIG. 8B). As shown in FIGS. 26A-26C, the pipette head220 can include a lever 221 that pivots upon contact with a portion ofthe container proximate to the used pipette rack 650. As shown in FIG.26C, the container sidewall 14 w or a member held thereon can be angledso that upon contact with the outer end of the lever 221, the lever 221pivots and pushes the used pipette tip 653 off the adapter 220 a andinto a receiving space in the rack 650. That is, as the pipette head 220moves downward toward the used rack 650, the lever 221 contacts the wall14 w, which forces one end of the lever upward and the end over thepipette tip 653 downward to push the used tip into the rack 650. Otherrelease configurations may also be used. For example, the used rack 650may be configured to restrict upward movement of the pipette tip 653 topull, push or otherwise force or strip the tip from the adapter 220 a.

The pipette head 220 and/or adapter 210 may attach to fluid lines andload cell wiring which can be elevated to clear the flexible barrier 16,and a flexible conduit back to the chassis 70. The load cell wiringcommunicates with a load sensor in communication with the sterile or newpipette tips. The load cell provides data used to control the loading ofnew pipettes onto adapter 220 a. For example, the robotic arm can causethe pipette head 220 to advance down with a force between about 5N toabout 50N to indicate the pipette tip 651 is properly attached. Adapter210 can also include a collar for locking and unlocking the adapter 210to the pipette head 220, e.g., as a non-limiting alternative to anengage/release handle mechanism.

Exemplary Flexible Barriers

FIGS. 9A-9E illustrate exemplary flexible barriers 16 according toembodiments of the present invention. Any suitable material can be usedfor the barrier. The barrier 16 can be substantially impermeable to air,moisture, and ethanol vapor. The barrier 16 may be moldable withoutlosing sufficient flexibility or impacting permeability. In someembodiments, the barrier 16 comprises a low-density polyethylene, butother alternatives could include a range of urethane materials. Inparticular embodiments, the flexible barrier is or comprises an“ARMORFLEX” material, available from ILC Dover Company, located inFrederica, Del.

The formed shape of the flexible barrier 16 can allow sufficient robotmovement and give the pipette head (or other internal interface) accessto the interior of the isolation container 12 without restriction orotherwise pinching or catching the flexible baggier. The shape of themolded flexible barrier 16 can be substantially self-supporting (e.g.,it will not collapse into the isolation container) once the flexiblebarrier 16 is sealed to the tray 14 forming the isolation container 12.

In some embodiments, a portion 910 of the barrier 16 may be adapted tointegrate with, receive, hold, attach to, mate with, seal with, support,or otherwise interact with a device for manipulating samples within thecontainer, such as, for example a pipette head 220, another fluidtransfer device, and/or another manipulation tool. The portion 910 canbe a substantially medial portion. The barrier 16 may include one ormore folds, pleats or undulations described as “convolutions” 920, someof which may be substantially concentric with others. FIG. 9Billustrates that the portion 910 may include at least one steppedportion 17, shown as a series of substantially vertically orientedstepped portions 17 ₁, 17 ₂, 17 ₃.

In some embodiments, as shown for example in FIGS. 9C and 9D, thebarrier 16 may include one or more radial convolutions 930. The radialand/or concentric convolutions may help facilitate a full range ofmovement of the pipette head (or other internal manipulation toolinterface), and help control the barrier shape during movement of thepipette head. A rim 940, flange or other feature may provide a surfacefor clamping, sealing, fixing, adhering, or otherwise attaching thebarrier 16 to the tray 14.

As shown in FIGS. 9C and 9D, the flexible barrier 16 can comprise aunitary sheet of material that is stretched or formed into a series ofsubstantially concentric folds, pleats or convolutions that can allowfor the desired movement of head 220. Outer perimeter regions of thebarrier sheet 16 t may have a different thickness than a center region16 c. In some particular embodiments, during fabrication, thickermaterial can be drawn down to the center during vacuum forming causingthinner central regions and thicker perimeter regions of the barriersheet 16. The center region 16 c may include a series of columnatedsteps 17 ₁-17 ₃, arranged smaller to larger as the barrier 16 moves awayfrom the workspace 18. In other embodiments, rather than a unitary sheetof material, the flexible barrier 16 may comprise a plurality ofco-joined sealed segments of the same or different suitable materials(not shown).

FIG. 9E illustrates the flexible barrier 16 sealably attached to a lowerportion of the pipette head 220 for subsequent engagement to the roboticarm 20. A robotic arm coupler 910 c resides outside the barrier 16. FIG.9F illustrates a cooperating coupler 210 c that is configured to engagethe coupler 910 c. As shown in FIG. 9G, fingers 211 are configured toenter apertures in the coupler body 910 a to releasably engage thebarrier 16. The assembly is shown in FIG. 9H (without the lowercontainer tray 14).

Exemplary Thermal Block Assemblies

As shown in FIG. 10, a nucleic acid processing system may incorporate athermal block assembly 1000 or any other temperature control device. Forexample, a nucleic acid processing system can incorporate a thermalcycler for synthesizing and amplifying nucleic acids. The thermal blockassembly 1000 may include, for example, a thermal (heater) block 30,which may be configured to receive a multiwell strip or plate such as aPCR plate 610, and a thermal cycler heated lid 1010 which may beactuated by a lid mechanism 310 to cover the PCR plate 610. For example,in some embodiments, the thermal cycler assembly is used for cDNAsynthesis, cDNA amplification, IVT RNA synthesis and DNA removal stepsof the process. Various types of thermal blocks and/or thermal cyclerdevices are available.

As shown in FIG. 10, the PCR plate 610 may be incorporated into theisolation container tray 14. In some embodiments, the perimeter of thePCR plate 610 can be sealed to an open (substantially rectangular)region formed in the base of the tray 14. Lower portions of the plate610 extend downwardly from the base of the tray to engage (typicallyreside in) a thermal cycler heater block 30 while the openings of wells611 in the plate 610 are accessible from inside of the sealed container12. The PCR plate 610 can be sealed to the isolation container tray 14so that the downwardly extending portions of wells 611 can be in directcontact with the thermal cycler or temperature control device.

In some embodiments, a compliant gasket, O-ring or other sealingmechanism may be used to attach the PCR plate 610 to the isolationcontainer tray 14. The compliant gasket may be attached with aheat-swaged clamping feature. In some embodiments, an over-moldedthermoplastic elastomer (TPE) seal 1030 is used for attaching andsealing the PCR plate 610 to the isolation container tray 14 as shown inFIG. 10. In other embodiments, PCR plate features may be formed, such asmolded and/or machined, into the isolation container tray substrate. Inother embodiments, a glue, tape and/or adhesive may be used to attachthe PCR plate 610 to the tray 14.

A lid, such as a heated thermal cycler lid 1010 may be used to reducethe loss of reaction fluid via evaporation. The lid 1010 may be lined,for example, with a flexible seal 430 to inhibit evaporated fluidescaping from some or each PCR well 611 and/or for maintaining isolationof the interior of the container 12. The lid 1010 can be heated toinhibit vapor condensing on the lid or any surface other than thereaction mixture. To provide access to all of the PCR wells, the thermalcycler lid 1010 may be lifted vertically to release the sealingpressure, then retracted substantially horizontally taking with it theflexible lid seal 430.

A drive system 310 for controlling the automatic movement of the lid1010 may be mounted to the work surface 200 of the processing system 10,as shown in FIG. 3. In other embodiments, the lid drive system 310 maybe mounted at other locations above or below the work surface 200.Referring now to FIG. 11, an exemplary lid drive system 310 is shownwith the lid 1010 in the retracted position. The arm 1122 is connectedto the lid 1010 and communicates with motor 1150; this provides formovement of the lid 1010 into and out of container 12, e.g., through aside-ingress/egress aperture 14 a in the wall of tray 14 where a lidseal (which can be described as a sleeve) is disposed between lid 1010and the interior of the container as shown in FIG. 12. Robotic tools ormanual input may also be via the seal 430.

FIG. 13A illustrates an exemplary configuration of the lid 1010 in thecontainer 12 and inside the chamber 430 c of the lid seal 430 whenextended. FIG. 13B illustrates the lid 1010 outside the container 12 ina retracted configuration.

The arm 1122 can be configured to direct the lid 1010 vertically as wellas horizontally. In the embodiment shown in FIGS. 11 and 12, the drivesystem 310 includes a vertical movement mechanism 1124 that provides forvertical movement of the lid 1010, e.g., when lid 1010 is positionedover the PCR plate 610 within the container 12. The vertical movementmechanism 1124 may provide a downward force sufficient to seal the lid1010 with the plate 610 and/or the block 30 (FIG. 10), for example,using one or more biasing members 1152. One or more motors 1150,actuators, gears, linkages or other electro-mechanical devices may beused to drive horizontal and/or vertical movement. As shown in FIG. 12,the drive system 310 includes an upper plate 1123 with a slot 1123 s.The arm 1122 includes a keel 1122 k that slides in the slot 1123 s. Thedrive system 310 also includes a lower plate 1126 that is attached tothe upper plate 1123 via pivots 1127 and biasing members 1152. Thevertical drive 1124 is attached to the upper and lower plates 1123, 1126and is configured to pull the upper plate 1123 down to force the lid1010 down once the lid 1010 is in the extended configuration inside thecontainer.

A portion of the drive system 310 can be physically attached to theinterior surface of the sleeve or lid seal 430 to facilitate the seal430 moving appropriately with the lid 1010. As shown, in FIG. 11, insome embodiments vacuum cups 1140 may be incorporated onto the lid 1010to engage the flexible lid seal 430 and to cause the seal 430 to remainin position during vertical and horizontal movement of the thermalcycler lid 1010. In other embodiments, the lid seal 430 may be attachedin other ways or may be sufficiently flexible to conform to the lid 1010and move in concert therewith.

In some embodiments, to facilitate a reliable seal between the isolationcontainer 12 and the flexible lid seal 430, a clamp plate or otherclamping member may be used to compress against an edge portion of theseal 430, such as via one or more sealing ribs (not shown) on the lidseal against the outside wall of the tray 14. In other embodiments, thelid seal 430 may be over-molded to the tray 14 using, for example, TPEas discussed above with respect to the PCR plate 610. O-rings, gaskets,adhesives, tapes, and mounting or sealing members may be used to providethe sealed connection of the lid seal 430 and the tray 14. FIG. 13Ashows the lid mechanism 310 with the lid 1010 and the seal 430 extendedinto the interior region of the tray 18 in an engaged position whileFIG. 13B shows the lid 1010 and retracted out through a wall ofcontainer 14. FIG. 13A illustrates the lid 1010 in position over athermal cycler and 96-well plate. FIG. 13C illustrates a partial sideperspective cutaway view of the lid mechanism 310 with the lid 1010extended into the interior region of the container 18.

Exemplary Waste and Elution Systems

Various purification means can be used to process samples, such asnucleic acids, proteins, cells, tissues, and the like. Such purificationdevices include, but are not limited to, magnetic beads, size exclusionmembranes, binding plates, filters, and binding columns. Such devicesare known to those of skill in the art.

In one embodiment, the processing system 10 uses a technique of elutingfrom binding columns for tumor total RNA isolation, cDNA purificationand IVT RNA purification. The binding column 1410 (FIG. 14) can containa silica membrane. A vacuum elution protocol can be integrated into theisolation container design. In other embodiments, a magnetic elutionprotocol can be integrated into the container.

One or more types/sizes of binding columns may be used. For example, insome embodiments, binding columns (e.g., Qiagen RNeasy™ columns) areused as they have a binding capacity suitable for the quantities of RNA,cDNA and IVT RNA that are processed. An additional, smaller Qiagen“Mini” column can be included to provide the ability to concentrate theRNA.

To inhibit the waste fluid from contaminating the elution vessels, thewaste and elution stations 632, 630, respectively, can be separate asshown for example in FIG. 6. In some embodiments, a manifold 50 is usedto hold the binding columns (see FIGS. 14 and 15). The manifold 50 canbe configured to hold one or more binding columns of varioussizes/types, e.g., Qiagen maxi-, midi- and/or mini-binding columns 1410.The manifold 50 may include wells 50 w for each binding column, liftingmembers or arms forming lift members 50 h for the pipette head 220 tolift it, and one or more slidably movable lids 1430 that can be used toclose off the unused binding columns. The lids 1430 can include a handle1430 h that can allow the head 220 to slide the lid 1430 in a desireddirection. In other embodiments, a manifold 50 may be configured to holdother types of substrates or devices to be used during a desiredprocess. The lift members 50 h can be configured as any suitable lift,including one or more handles (as illustrated), wings, flanges, or otherfeatures to facilitate movement of the manifold 50 between stations.

FIG. 15 shows an example of a mechanism and method for moving a manifold50 between two or more stations or area of the container 12, e.g.,between the waste and elution stations 632, 630, respectively. Thepipette head 220 and robot 20 may be used to move the binding columnmanifold 50 and the columns 1410 to and from, i.e., between the twostations. In the embodiment shown, the manifold 50 may include handles50 h, wings or other features that interface with corresponding features520 on the pipette head 220, and are used to simplify the engagementbetween the robot 20 and the binding manifold 50.

Each isolation and purification process can use a different set ofcolumns; the pipette head robot may be used to reposition the lids 1430to close the appropriate set of unused columns. This reduces orminimizes the airflow through the manifold 50, allowing the pump togenerate a sufficiently high vacuum during the isolation and/orpurification processing steps. To reduce operator handling of thebinding columns 1410 after sterilization, the columns 1410 may be loadedinto the manifold 50, and the assembly sealed into suitable packagingand gamma-sterilized in the manifold 50. At the time of loading theisolation container at a use and/or assembly facility (such as in asuitable clean room), an operator can remove the packaging from themanifold 50 and place it into the waste station 632, without everneeding to directly handle the binding columns.

The waste and elution station configurations can be flexible and/orscalable. The manifold 50 design can be large enough to allowmodification or use to suit a 96-well format, or any other type ofplate, well or columns, or changes in the combination of maxi-, midi-and mini-columns 1410.

Referring now to FIGS. 16 and 17, the elution station 630 can include anelution tray 1610 configured to capture the final output of the bindingcolumns (e.g., fluid containing DNA, RNA, etc.). To inhibit and/orprevent possible cross contamination between DNA and RNA, each bindingcolumn 1410 can have its own receptacle 1612 below it in the elutiontray 1610. In some embodiments, the elution process employs a highvacuum, which may generate a significant airflow through the bindingcolumn(s) 1410. The elution receptacles 1612 may be shaped to allowsplashed fluids, if any, to run off and pool in a collection vesselwhile deflecting the air stream away from the pooled fluid. Additionalfeatures may reduce or minimize the airflow that passes from onereceptacle over another. Further discussion of the tray 1610 will beprovided with respect to FIGS. 29A-29C.

Waste station 632 may include a waste tray 1630 and waste tray cover1640. A waste tray cover 1640 may be incorporated into waste tray 1630to minimize the evaporation of the waste fluid contained in the wastestation 632. The waste tray cover 1640 may be shaped to direct all wastefluid to flow down into one or more (shown as a central) drain hole 1641where it is protected from the main airflow below the cover 1640 insidethe waste station 632. FIGS. 30A and 30B illustrate another embodimentof the waste tray cover 1640 which employs a plurality of waste drainholes 1641 using a tray cover that is shaped substantially similar tothe elution tray 1610. In the shielded environment below the waste traycover 1640, the air directly above the waste fluid can maintain a highhumidity, thus reducing further evaporation. Further discussion of thetray cover 1640 is provided with respect to FIGS. 30A and 30B.

In some embodiments, a vacuum system 1600 circulates air throughcontainer 12. In some embodiments, the isolation container vacuum system1600 is configured to eliminate the need for filters. The vacuum systemcan comprise tubing 1650, typically flexible tubing, connected from apump head 1602, such as via a pinch valve 330 (FIG. 17), to the waste632 and elution 630 stations. The outlet of the pump head returns theaspirated air back into the isolation container 12.

FIG. 17 shows a circulation path according to one embodiment of thevacuum system 1600. In this embodiment, the tubing 1650 and disposablepump head 1602 may be attached to the isolation container tray 14 duringmanufacture. These can be sterilized and shipped as part of the assemblyassociated with the isolation container tray 14. Once an isolationcontainer 12 is loaded and sealed, the vacuum system 1600 can remainclosed throughout the rest of the processing. During installation of anisolation container into the processing system 10, the vacuum tubing1650 can be inserted into the pinch valve 330 and the disposable pumphead 1602 is connected to the vacuum pump 320. In some embodiments, thepump 320 only operates when vacuum is required. The pinch valves 330 maybe used to direct the vacuum to the appropriate station. When processingis completed, the tubing 1650 may be extracted from the valve 330 andthe pump head 1602 is disconnected from the pump 320. The tubing 1650and pump head 1602 can be single-use disposable, along with the rest ofthe isolation container 12.

FIG. 18 shows a cutaway view of a tray 14 in place on the work surface200 of a processing system 10, showing exemplary locations of the pinchvalve 330 and disposable pump head 1602. Peltier plates 370 and 372 mayprovide cooling for a segment of the rear wall 14 p of the tray 14 andfor the waste station catchment in the base of the tray 14,respectively. The wall segment 14 p may be substantially planar(typically substantially vertical, but may be angled) and project inwardto define a receiving space for the plate 372. The wall 14 p may beconfigured to cooperate with the plate 372 to define a condensationwall. It is noted that in FIG. 18, the thermal cycler lid drive system310 is shown with the lid extended into container, without the seal 430in place and the disposable pump head 1602 is shown, without theflexible vacuum pump tubing 1650, in place.

Exemplary Process Control and/or Yield Measurement Systems

In some embodiments, disposable cuvettes 660, 2060 can be used, forexample, as shown in FIG. 19A and FIG. 20F. One or more cuvettes 660,2060 may be fixedly or releasably mounted to isolation container tray14, e.g., such that cuvettes 660, 2060 extend vertically down from thebottom of tray 14.

In some embodiments, the concentration and/or purity of samples, e.g.nucleic acids or proteins, can be measured using a spectrophotometer1900 s (FIGS. 3, 19B) and cuvettes 660 (FIGS. 19A, 20F) or alternativeoptical methods for concentration measurements, e.g., fluorometer, canbe similarly incorporated in the system.

In some embodiments, as shown in FIG. 19A, one or more disposableconcentration cuvettes 660 are sealably mounted to the tray 14 via aflexible sealing member 1918, such as a gasket or O-ring. The tray 14includes mounting members 14 m that engage a clamp 1920 to hold thecuvette 660 tightly against the sealing member 1918, thus sealing thecuvette 660 to the tray 14 and maintaining the sealed processingenvironment inside the isolation container. As shown, the cuvette 660can include an outer edge that is formed as a rigid upper lip 661. Theclamp 1920 includes an inner upstanding arm 1921 that resides againstthe lip 1901 and an outer arm 1922 that engages the mounting member 14 mto force the cuvette 660 against the tray 14. The cuvette 660 includes acuvette measurement window 660 w, typically at a lower portion thereof.

Alternatively, the cuvettes 660 (or cuvettes 2060) can be molded intothe tray, overmolded, mechanically clamped, solvent bonded, adhesivelyaffixed, or the like. The spectrophotometer 1900 s (FIG. 3) can bemounted on the processing system 10, e.g., beneath the isolationcontainer 12. The spectrophotometer 1900 s can be configured to makeabsorbance measurements through cuvettes 660 (FIG. 20F) while remainingoutside the isolation container.

In some embodiments, there is a cuvette 660, 2060 for each measurement,such that no cuvette need be reused. The processing system 10 can beconfigured to move a spectrophotometer cuvette holder 360 (see, e.g.,FIG. 3, 19B) from cuvette 660 to 660 for each measurement as required.In some embodiments, cuvettes 660 and/or 2060 may be removed from thetray 14, washed and reused in subsequent trays 14. In other embodiments,removable cuvettes 660, 2060 are single-use only (i.e., single-usedisposable).

Various types of spectrophotometers are known and may be utilized fornucleic acid concentration and purity measurements. Thespectrophotometer 1900 s may be able to communicate with and becontrolled by a controller associated with the processing system 10. Forexample, the processing system 10 can command the spectrophotometer 1900s to take a measurement and the spectrophotometer 1900 s can be able toreturn the absorbance measurements, typically at ultravioletwavelengths, such as for example, at about 260 and/or 280 nm, or at anyother wavelength of interest. Additionally, the geometry of thespectrophotometer 1900 s and particularly that of a cuvette holder 360can be integrated with other components of the overall system.

FIG. 19B illustrates a spectrophotometer head mechanism that is used tomove a single detector head between a number of cuvettes 660, shown as(4) cuvettes 660 in FIG. 20F. A robotic operational platform may be usedto position the spectrophotometer cuvette holder 360, e.g., to raise,lower and traverse between cuvettes 660. The spectrophotometer 1900 sand light source may be mounted in an electrical cabinet of the system,e.g., on a left or right side of the system. Optical fibers may be usedto allow the cuvette holder 360 to move while the spectrophotometer 1900s and light source remain stationary. That is, as shown in FIG. 19B, aspectrophotometer drive mechanism can be used to move a single detectorhead 1900 h to serially hold each of the different cuvettes 660. Thedetector in the detector head 1900 h can be coupled to thespectrophotometer 1900 s via a plurality (typically two) optical fibers.In other embodiments, each cuvette 660 could be located in an individualcuvette holder 360 and the light source switched between the opticalfibers connecting the light source to the multiple cuvette holders (notshown) to communicate with the spectrometer for measurements.

Volume measurement(s) of a sample may be used in combination with theconcentration measurement(s) to determine yields. In some embodiments, avolume measurement unit 2000 utilizes the pipetting system and a fluidsensor in a dedicated vessel, e.g., as shown in FIGS. 20A-20E. Forexample:

-   -   FIG. 20A shows an empty cuvette 2060 in a detector head 2002,        wherein light from two IR emitters 2010 does not pass through        the cuvette 2060, and is received by two IR receivers 2020 for        reflected light.    -   When a fluid 2040 to be measured is pooled in the volumetric        cuvette 2060, e.g., as shown in FIG. 20B, light from the two IR        emitters 2010 is transmitted through the cuvette 2060 and the        fluid 2040 and is not received by the two IR receivers 2020 for        reflected light.    -   While a control system monitors the presence of the fluid        detected by the system 2000 at the bottom of the measurement        vessel 2060, the pipette head 220 is used to aspirate the sample        2040 into a pipette tip 651 as shown in FIG. 20C.    -   As shown in FIG. 20D, when the sensor system 2000 reports fluid        absent, the syringe pump 350 (FIG. 3) can be stopped and the        volume aspirated can be electronically determined and recorded.        Thus, the cuvette 2060 has a prism design in the bottom. Using a        light source with detectors the instrument can determine the        presence or absence of liquid in the cuvette 2060 based on the        reflection. This, in combination with the pipetting device        determines the volume by measuring the liquid it is removing        until receiving the signal that no liquid is present. FIG. 20E        illustrates that the system 2000 can employ a plurality of        spaced apart detector blocks 2002, each having its own pair of        transmitters 2010. Each block 2002 can be configured to hold and        cooperate with a respective cuvette 2060 for obtaining a volume        measurement using the volume measurement system 2000.

As shown in FIG. 20F, the tray 14 can include a plurality of both thevolume cuvettes 2060 (e.g., volume measurement cuvette) and a pluralityof spectrophotometer cuvettes 660 (the cuvettes 660 can be used forconcentration measurements). The spectrophotometer cuvette 660 can be astandard UV visible plastic disposable such as BioRad “trUViewcuvettes”. The device 10 can include spectrophotometer components formeasurements. Suitable spectrophotometer components can be obtained fromOcean Optics, having a place of business at Dunedin, Fla.

Exemplary Process Constituent Storage Systems

In some embodiments, reagent vessels are used to contain the reagents,the sample and mixing volumes of fluid combinations within the isolationcontainer 12. Reagent vessels with various different capacities may beused to accommodate the range of fluid volumes used during the process.

To reduce the risk of reagent contamination, evaporation or spillage,each vessel that contains reagent can be sealed, typically over a topsurface, such as with polypropylene-lined foil, prior to loading intothe tray 14. The seal can be configured to be pierced by a pipette tip651. The seal allows reagents to be loaded and sealed into theappropriate reagent vessel and stored without the risk of contamination,spillage or evaporation.

An example of a reagent rack 620 is shown in FIG. 21A. Rack 620 may haveany desired layout, can accommodate any reagent vessel size, and can beadapted for any process. In the example shown, the reagent tray or rack620 includes a base 2100 for housing a number of vessels 2110, 2112,2114, typically tubes, of different sizes that may contain for examplesample and reagents, as well as mixing vessels. Each of the vessels canbe supported within the base 2100, and a cover 2120 may be clipped orotherwise attached onto the rack 620 to ensure the vessels are keptsecurely in place. The cover 2120 includes apertures to allow thepipette tip to access the vessels held thereunder.

In some embodiments, reagents, samples, or other fluids can be loadedinto the rack 620 as a sub-assembly, and the rack 620 is then loadedinto the tray 14. In some embodiments, the design of the reagent rack620 is such that it can only be placed into the isolation container trayin one orientation. Alternatively, the reagent rack 620 may be molded orotherwise made an integral part of the isolation container tray 14.Reagents may be loaded directly into an integral reagent tray or tubesor other vessels containing a reagent may be placed in the reagent tray.The placement of the vessels 2110, 2112, 2114 at a defined rack location(address) allows the robotic interface to automatically engage thecorrect vessel (reagent) at the correct time in the process.

In some embodiments, the tray layout allows access to all the vesselswithout the pipette tip having to pass over other dissimilar reagents,and thus reduces the chance of reagent cross contamination. Of course,other configurations may be used, for example, depending upon thedesired processes to be performed.

FIG. 21B illustrates that a multi-point (shown as a three-point)piercing sequence can be used to open the seal on a vessel 2110, 2112,2114, respectively, before withdrawing the (reagent) liquid. That is,the pipette 651 can be directed to pierce a seal covering at two outerlocations before moving to the actual withdraw piercing (typically at acenter location) to facilitate pipetting accuracy.

Exemplary Aliquot Removal Systems

During automated processing in system 10, one or more aliquots of asample and/or products may be used for process evaluation, control,quality testing and/or storage for further processing (e.g., seealiquots tubes 440 in FIGS. 4 and 5).

In some embodiments, the isolation container 12 and processing system 10allow aliquot removal during processing without compromising the sealednature of the isolation container 12. A plurality of flexible tubes canbe in fluid communication with an interior sample capture region of thecontainer 12 and extend from the system 10 to be externally accessibleby an operator. Each flexible tube is presealed or has a closed endportion. Each aliquot may be aspirated into an individually labeledcontainer 440, for example a tube, while the container 12 remainsclosed. An operator or an automated sealer may seal the aliquot into thetube 440, e.g., upstream of the captured aliquot of fluid, such as byusing an RF tube sealer, or other seal closure mechanism. Once sealed,an aliquot tube can be detached from the isolation container and takenaway for storage, testing, or further processing.

In some embodiments, an aliquot assembly 40 is used for aspirating thealiquot fluid into the tube 440 and utilizes a clamping member and theelasticity of aliquot tubing 440 to draw the fluid from inside thecontainer 12 into the tube. This mechanism can be automated tofacilitate the tubing being clamped for a short time, thus reducing thelikelihood of any change in the elasticity of the tubing caused byclamping deformation.

FIG. 22 illustrates an exemplary method 2200 of using an aliquotassembly 40, and shows a series of steps (from left to right and top tobottom) that can be used to load the aliquot tubes 440 with an aliquotfluid 445. As shown, an aliquot tube 440 with a closed end 441 issealably attached to the tray 14 under a well 14 w. In this example, aclamping member (tube expressor) 2210 is pressed against one or morealiquot tubes 440 to expel air from the tubes (as shown by the secondand third figures). After or while air is discharged in response to thetube being completely compressed by clamp 2210 (with upper clampingmember 2211 forced down against the tube and other clamp surface), thealiquot substance 445 is placed or dispensed in the well 14 w incommunication with the tube 440. Upon subsequent release of the clampingforce, an aliquot of fluid 445 is drawn into the tube 440 by a negativepressure within the elastic tube. A tube sealer 2220 (such as an RFwelder) may be used to seal (at 442) and optionally cut the aliquot tubeupstream of the seal to isolate the sample 445 within the tube. Othersealant mechanisms may also be used.

Exemplary Methods of Assembling a Container

FIG. 23A is an exploded representation of a kit 2500 that can beprovided to allow an operator to prepare a closed system for processing.The kit 2500 can include the tray 14, the binding manifold 50, thepipette tip rack 650 (and pipette tips 651), and cuvettes 2060, 660. Thetray 14 can be packaged and shipped to a use site with the aliquot tubes440 attached and/or the vacuum head and associated tubing attached. Thecomponents can be sterilized and packaged (or packaged, then sterilized)in a sterile package for shipment and handling. The components may besurface sterilized at a point of use and the assembly of the componentsmay be in a Class 10,000, 1000, or even 100 clean room. The componentsmay also be provided separately or preloaded in other combinations.

FIG. 23B is a partially assembled view of the kit shown in FIG. 23A.FIG. 23C is a bottom view of the partially assembled kit shown in FIG.23B. FIG. 23D is a top view of the container 14 shown in FIG. 23A priorto assembly of the discrete components shipped with or separate from thecontainer or tray 14.

FIG. 24 illustrates an exemplary method of sealing, respectively, anisolation container 12 according to embodiments of the invention (forexample, for aseptic processing nucleic acids from one or morebiological samples). For such aseptic processing, the barrier 16 andtray 14 sections of the isolation container assembly 12 may beindividually pre-packaged and sterilized. When the isolation container12 is needed for processing, the tray 14 and barrier 16 may betransferred to a biological safety cabinet or equivalent clean,controlled area for loading.

As shown in FIG. 23A, the tray 14 of an isolation container 12 may beloaded with one or more reagent trays 620 comprising samples andreagents to be used in the desired process. The assembly and/or loadingof the tray 14 can take place with the tray 14 on the trolley 399 (FIG.27) as discussed above. One or more pipette tip racks 650 and bindingcolumn manifolds 50 may also be loaded into the working area 18 of thetray 14. In some embodiments, one or more cuvettes 660, 2060 areattached to the base of tray, for example, as shown in FIG. 20F.Depending upon the desired assay or process to be performed, othersamples, analytes, reagents, vessels, other consumables, devices, toolsand/or other items may be loaded into tray 14.

In some embodiments, after all of the desired items are placed within orattached to the tray 14, the barrier 16 can be attached to the tray 14to seal the assembled isolation container 12, for example as shown inFIG. 24. In some embodiments, an O-ring 2400 may be used to seal theflexible barrier 16 to the tray 14. The O-ring can be forced snugly overthe barrier 16 and into the perimeter slot on the tray 14. In otherembodiments, one or more gaskets, clips, clamps, fasteners, adhesives,and/or other attachment means may be used.

As shown in FIG. 27, the tray 14 can be locked to a holding surface on atrolley during transport. After the tray 14 is loaded and sealed withthe barrier 16, the isolation container 12 may then be transferred tothe work surface 200 of the processing system 10. Alternatively, theisolation container can be assembled and sterilized. Then, reagents andsamples can be introduced into a sealed isolation container usingstandard aseptic transfer methods or by other closed means. The trolleycan also be used to hold the tray 14 during assembly. Alternatively, aseparate barrier isolator trolley-assembly cart may be used whenassembling the isolation container 12 (not shown). Such a barrierisolator trolley may be integrated into the barrier isolator itself andcan mate with the transport trolley as discussed above.

The isolation container assembly 12 may be installed into the system 10and connected to each piece of equipment used for processing thesamples. To ensure correct alignment, the rigid tray 14 may be clampedonto the work surface 200. The flexible barrier 16 and pipette head 220may connect to the robotic pipette head adapter 210 (see e.g., FIG. 2).As shown for example, in FIG. 12, thermal cycler evaporative seal 430may connect to the thermal cycler lid 1010, e.g., using vacuum cups 1140or other fasteners or attachment means, and vacuum tubing and thedisposable pump head for the waste and elution stations are fitted tothe corresponding valves and pump. To remove an isolation container, thereverse procedure may be used.

FIG. 25 is a flow chart of operations that can be carried out at amanufacturing site according to embodiments of the invention. As shown,an input sample 3000 is provided (typically from a patient collectionsite or a subject's specimen storage site). When time-sensitive, thespecimen can be shipped for receipt at the manufacturing site within12-48 hours (such as overnight). At the manufacturing site, theisolation container 12 is prepared in a barrier isolator (block 3010)and sample may also be prepared (block 3000 p) prior to placement in thecontainer (e.g., homogenized, mixed and/or centrifuged). The outerpackaging of the isolation container components, e.g., the tray 14, thebarrier 16 and the container contents can be sterilized (block 3002).The assembled and sealed isolation container can be assembled (block3004) and integrity-tested (block 3006) to confirm a sealed state.

The closed assembled isolation container can be moved to the instrumentfor processing (block 3020). As shown, the RNA from the patient samplecan be isolated (block 3022) using silica membrane columns andappropriate RNA binding and elution reagents. Concentration and volumedeterminations are performed out to calculate yields and theconcentration is normalized (block 3024). The cDNA is synthesized by areverse transcription (RT) reaction, amplified through PCR, and purified(block 3026) using silica membrane columns and appropriate cDNA bindingand elution reagents. Concentration and volume determinations areperformed to calculate yield and the concentration is normalized (block3028). IVT RNA is produced from the resulting cDNA, treated with DNaseand purified (block 3030) using silica membrane columns and appropriateRNA binding and elution reagents. Concentration and volumedeterminations are performed to calculate yield and the concentration isnormalized (block 3032) resulting in the final amplified RNA (block3034). The RNA disposable can be integrity-checked (block 3036) toconfirm that the system is still sealed and isolated from theenvironment to thereby ensure that there was no contamination during theprocess. Intermediate and/or final products can be output as one or morealiquot amounts (block 3050).

FIGS. 29A-29C show an exemplary elution tray 1610. As shown, thereceptacles 1612 are spaced apart and angled to inhibit splash-overduring use of a respective receptacle 1612. Two or more receptacles 1612reside on each side of an upwardly extending barrier 1614 and theprimary tubular receiving portion 1615 merges into a shallow channel1616 that tapers downwardly and becomes deeper and narrower as it movestoward the tubular body portion 1615. The forward portion of the channel1616 is also tapered inwardly and downwardly to capture splash and todirect the liquid to flow to the primary tubular portion 1615.

FIGS. 30A-30B illustrate a similar configuration for the waste traycover 1640 but the tips 1632 of the receptacles are open or haveapertures for draining the waste to the waste station 632 (FIG. 16).

FIG. 31 is a schematic illustration of a control circuit 4000 that is incommunication with sensors 4010, 4020, 4030 that can be used to monitorthe status of the inside of the container or provide desired feedback toallow the automatic control of components (heaters, coolers, robotic armmovement and the like). As shown, the control circuit 4000 can be incommunication with the user interface 61 (FIG. 1). The sensors caninclude a load sensor 4010, a temperature sensor 4020, and a pressuresensor 4030. The load sensor 4010 can be in communication with the newpipette rack to provide loading force data used to control theengagement force applied by the robotic arm to engage a new pipette. Thetemperature sensor 4020 can provide temperature data regarding theinternal environment or a particular location within the closedcontainer environment (e.g., one or more of the workstations). Thepressure sensor 4030 can be used to monitor the sealed integrity statusof the closed environment and/or vacuum during isolation or purificationprocedures. Multiple sensors of each or some of the sensors can be usedand other sensors may also be used. The control circuit 4000 may residewholly or partially in the system 10 or wholly or partially with or onthe container 12.

As will be appreciated by one of skill in the art, embodiments of theinvention may be embodied as a method, system, data processing system,or computer program product. Accordingly, the present invention may takethe form of an entirely software embodiment or an embodiment combiningsoftware and hardware aspects, all generally referred to herein as a“circuit” or “module.” Furthermore, the present invention may take theform of or use a computer program product on a computer-usable storagemedium having computer-usable program code embodied in the medium. Anysuitable computer readable medium may be utilized including hard disks,CD-ROMs, optical storage devices, a transmission media such as thosesupporting the Internet or an intranet, or magnetic or other electronicstorage devices.

Computer program code for carrying out operations of the presentinvention may be written in PLC code such as Graph, Ladder or SCL.However, the computer code can be alternatively or additionally writtenin an object oriented programming language such as Java, Smalltalk orC++ and/or conventional procedural programming languages, such as the“C” programming language or in a visually oriented programmingenvironment, such as VisualBasic.

Certain portions (or all) of the program code may execute entirely onone or more of the system's computer(s), partly on the systemcomputer(s), as a stand-alone software package, partly on the systemcomputer(s) and partly on a remote computer or entirely on the remotecomputer. In the latter scenario, the remote computer may be connectedto the system computer through a local area network (LAN) or a wide areanetwork (WAN), or the connection may be made to an external computer(for example, through the Internet using an Internet Service Provider).In some embodiments, some program code may execute on local computersand some program code may execute on one or more local and/or remoteserver. The communication can be done in real time or near real time oroff-line using a volume data set provided from the imaging modality.

The invention is described in part with reference to flowchartillustrations and/or block diagrams of methods, systems, computerprogram products and data and/or system architecture structuresaccording to embodiments of the invention. It will be understood thateach block of the illustrations, and/or combinations of blocks, can beimplemented by computer program instructions. These computer programinstructions may be provided to a processor of a general-purposecomputer, special purpose computer, or other programmable dataprocessing apparatus to produce a machine, such that the instructions,which execute via the processor of the computer or other programmabledata processing apparatus, create means for implementing thefunctions/acts specified in the block or blocks.

These computer program instructions may also be stored in acomputer-readable memory or storage that can direct a computer or otherprogrammable data processing apparatus to function in a particularmanner, such that the instructions stored in the computer-readablememory or storage produce an article of manufacture includinginstruction means which implement the function/act specified in theblock or blocks.

The computer program instructions may also be loaded onto a computer orother programmable data processing apparatus to cause a series ofoperational steps to be performed on the computer or other programmableapparatus to produce a computer implemented process such that theinstructions which execute on the computer or other programmableapparatus provide steps for implementing the functions/acts specified inthe block or blocks.

As illustrated in FIG. 32, embodiments of the invention may beconfigured as a data processing system 4116, which can be used to carryout or direct operations of the rendering, and can include a processorcircuit 4100, a memory 4136 and input/output circuits 4146. The dataprocessing system may be incorporated in, for example, one or more of aprogrammable logic controller (PLC), personal computer, workstation,server, router or the like. The system 4116 can reside on one machine orbetween a plurality of machines. The processor 4100 communicates withthe memory 4136 via an address/data bus 4148 and communicates with theinput/output circuits 4146 via an address/data bus 4149. Theinput/output circuits 4146 can be used to transfer information betweenthe memory (memory and/or storage media) 4136 and another computersystem or a network using, for example, an Internet protocol (IP)connection. These components may be conventional components such asthose used in many conventional data processing systems, which may beconfigured to operate as described herein.

In particular, the processor 4100 can be commercially available orcustom microprocessor, microcontroller, digital signal processor or thelike. The memory 4136 may include any memory devices and/or storagemedia containing the software and data used to implement thefunctionality circuits or modules used in accordance with embodiments ofthe present invention. The memory 4136 can include, but is not limitedto, the following types of devices: ROM, PROM, EPROM, EEPROM, flashmemory, SRAM, DRAM and magnetic disk. In some embodiments of the presentinvention, the memory 4136 may be a content addressable memory (CAM).

As further illustrated in FIG. 32, the memory (and/or storage media)4136 may include several categories of software and data used in thedata processing system: an operating system 4152; application programs4154; input/output device drivers 4158; and data 1456. As will beappreciated by those of skill in the art, the operating system 4152 maybe any operating system suitable for use with a data processing system,such as IBM®, OS/2®, AIX® or zOS® operating systems or Microsoft®Windows®95, Windows98, Windows2000, WindowsXP or WindowsCE operatingsystems, Unix or Linux™. IBM, OS/2, AIX and zOS are trademarks ofInternational Business Machines Corporation in the United States, othercountries, or both while Linux is a trademark of Linus Torvalds in theUnited States, other countries, or both. Microsoft and Windows aretrademarks of Microsoft Corporation in the United States, othercountries, or both. The input/output device drivers 4158 typicallyinclude software routines accessed through the operating system 4152 bythe application programs 4154 to communicate with devices such as theinput/output circuits 4146 and certain memory 4136 components. Theapplication programs 4154 are illustrative of the programs thatimplement the various features of the circuits and modules according tosome embodiments of the present invention. Finally, the data 4156represents the static and dynamic data used by the application programs4154 the operating system 4152 the input/output device drivers 4158 andother software programs that may reside in the memory 4136.

The data 4156 may include (substantially real-time, archived or stored)Process Sequence data sets 4126 and/or closed environment sensorfeedback data 4127. As further illustrated in FIG. 32, according to someembodiments of the present invention application programs 4154 includean Automated Process Sequence and Monitoring Module 4125. Theapplication program 4154 may be located in a local server (or processor)and/or database or a remote server (or processor) and/or database, orcombinations of local and remote databases and/or servers.

While the present invention is illustrated with reference to theapplication programs 4154 in FIG. 32, as will be appreciated by those ofskill in the art, other configurations fall within the scope of thepresent invention. For example, rather than being application programs4154, these circuits and modules may also be incorporated into theoperating system 4152 or other such logical division of the dataprocessing system. Furthermore, while the application program 4154 isillustrated in a single data processing system, as will be appreciatedby those of skill in the art, such functionality may be distributedacross one or more data processing systems in, for example, the type ofclient/server arrangement described above. Thus, the present inventionshould not be construed as limited to the configurations illustrated inFIG. 32, as it may be provided by other arrangements and/or divisions offunctions between data processing systems. For example, although FIG. 32is illustrated as having various circuits and modules, one or more ofthese circuits or modules may be combined or separated without departingfrom the scope of the present invention.

Other Embodiments

The foregoing exemplary system 10 was provided to illustrate variousaspects and features of the present invention, and is not intended tolimit the scope of the present invention to systems, devices and methodsfor nucleic acid processing. Rather, one skilled in the art willappreciate that various other applications fall within the scope of thepresent invention, including, for example, systems and methods forfabricating, assembling, processing or otherwise manipulating any items,typically in a closed container. The following sections provideadditional examples of applications in which the apparatus and methodsof the present invention may be employed.

Examples of Other Biological and/or Pharmaceutical Applications

In other aspects, the present invention provides methods of transferringa material, for example, a fluid, from a source to a target in a closedenvironment, wherein the closed environment is defined as the interiorregion or chamber in the container of an apparatus as described in thepresent application. A fluid to be transferred can contain, for example,a biologic; drug; toxin; isolate; radioisotope; virus; bacteria;eukaryotic cell; extract; analyte; biological specimen such as, forexample, blood, plasma, saliva, etc.; vaccine; nucleic acid; protein;foodstuff; and the like, including suspensions, mixtures, and so on,thereof.

Applications for the instant systems and methods can be found, forexample, where contamination of the material to be transferred insidethe closed environment is to be avoided by substances potentially in theenvironment outside of container. In certain embodiments, the systemsand methods can be directed to preparation of materials for, or forprocessing materials in, diagnostic assays, such as, by way ofnon-limiting examples, sterility assays, forensic analyses, or qualitycontrol assays, for example, to test purity of biologics to be used inclinical applications. In other embodiments, the instant methods can befor the preparation or manufacture of drugs and biologics, such asvaccines, and nucleic acids for experimental and/or clinical use.Various assays and methodologies that may be suitable for use in thesystems and methods of the present invention are known to those of skillin the art as described, for example, in United States Pharmacopeia,United States Pharmacopeia and National Formulary (USP 29-NF 24) (2006),U.S. Department of Health and Human Services, Guidance for Industry:Sterile Drug Products Produced by Aseptic Processing—Current GoodManufacturing Practice (September 2004), each of which is incorporatedby reference herein in its entirety.

It will be understood by those skilled in the art that a target to whicha fluid is transferred can be any receptacle, such as, for example, atube, vial, vessel, and the like, that is intended to hold thetransferred fluid. In some embodiments, the target is a substrate, suchas a binding column, size exclusion column, chromatography media, orfilter, or the like. However, depending upon the particular applicationin the methods provided, any flow-through device, that is, any deviceintended to capture and/or separate and/or concentrate one or morecomponents of the transferred fluid from another component of the fluid,can be used.

FIG. 14 provides an example of a configuration of an exemplary apparatuswherein binding columns 1410 situated in the closed environment are atarget for transferred fluid containing, for example, nucleic acids. Inother embodiments, the manifold described in FIG. 14 can be adapted tofit filters or other devices discussed above.

In some embodiments, systems and methods provided by the invention canallow for aseptic preparation of an amplified nucleic acid product,comprising the steps of isolating RNA from a cell extract and amplifyinga nucleic acid product from at least one RNA molecule from the isolatedRNA, wherein the isolating and amplifying steps are performed in aclosed environment as defined as the interior region or chamber in thecontainer of an apparatus as described in the present application,thereby aseptically preparing the amplified nucleic acid product.

In certain embodiments for the aseptic preparation of an amplifiednucleic acid product, the amplified nucleic acid product is derived froma pathogen, such as from a viral pathogen. In certain embodiments, theamplified nucleic acid product is derived from human tumor RNA. Anamplified nucleic acid product from a human tumor RNA can be used, forexample, in the preparation of a vaccine or immunotherapy.

The instant methods can be used for the sterilization of a material, forexample, a biologic as defined under Title 21 of the United States Codeof Federal Regulations, nucleic acid, and/or protein, and so forth,under aseptic conditions. Guidelines regarding the preparation of suchmaterials for clinical applications are described in, e.g., U.S.Department of Health and Human Services, Guidance for Industry: SterileDrug Products Produced by Aseptic Processing—Current Good ManufacturingPractice (September 2004).

Thus, in certain embodiments, methods are provided for the sterilizationof a material comprising transferring a fluid comprising the materialfrom a source to a filter and collecting the material after passagethrough the filter into a receptacle, wherein the source, filter andreceptacle are contained in a closed environment as defined as theinterior chamber or region in the container of an apparatus as describedin the present application. Filters for sterilization are commerciallyavailable, for example, from Whatman Inc. (Florham Park, N.J.).

It will also be recognized that transferring material, for example,dispensing the material into a vial, can be hazardous if the materialcomprises a pathogen, such as is the HIV virus, or other certainmicroorganisms, or comprises a biological toxin or a radioactivesubstance. Transferring such materials into receptacles in a closedenvironment such as defined in embodiments of the present invention canbe used to reduce the potential exposure of the hazardous material tothe individuals undertaking the transfer. Thus, in certain embodiments,methods are provided wherein a hazardous material is transferred from asource to a target in a closed environment as in the container of anapparatus as described in the present application. These methods can beapplied to a variety of procedures and assays where hazardous materialsare transferred between a source and a target, as will be known to thoseof skill in the art.

The apparatus of the present invention can be used for otherapplications typical of protein purification, nucleic acid purification,and other molecular biology processes. Such applications include, butare not limited to, cell lysing (e.g., mechanical, chaotropic, thermal,or enzymatic lysing of cells), macromolecule purification (e.g., ionexchange, bead, molecular weight cutoff), and recovery (silica elution,bead elution, membrane elution).

Exemplary Lithography, Microfabrication and Related Applications

Embodiments of the present invention can be used in any situation whereclean room conditions are used or isolation is desired, such as, forexample, lithography and assembly processes. In some embodiments, theapparatus of the present invention satisfy the conditions of a class 1,class 10, class 100, class 1000, class 10,000, or class 100,000 cleanroom as set forth by the U.S. Federal Standard 209b for clean roomclassification. See, Federal Standard No. 209B 1992, “Clean Room andWork Station Requirements, Controlled Environment,” dated Apr. 24, 1973,which is hereby incorporated by reference in its entirety. As such,embodiments of the present invention can be used for a broad spectrum ofprocesses that use a clean room environment. Such processes includelithography processes such as, but not limited to, wafer patterning(also known as photomasking, masking, photolithography,microlithography), doping (e.g., thermal diffusion, ion implantation),heat treatment (e.g., thermal, radiation). Specific patterning processesthat can be conducted in the apparatus of the present invention include,but are not limited to (i) resist application (positive or negative);(ii) exposure (e.g. by contact, proximity, scanning projection, andstepper) to high pressure mercury, X-rays, or E-beams; (iii) imaging(e.g., single layer resist, multilayer resist, application ofantireflector layers, off-axis illumination, planarization, contrastenhancement; and (iv) etch (e.g., wet chemistry-liquid/vapor, dry,plasma, lift-off, ion milling, reactive ion etching. Specific heattreatment processes include, but are not limited to, hot plate,convection, rapid thermal processing (RTP), and infrared. Moredescription of these lithography processes are described in Van Zant,Microchip Fabrication, Fourth Edition, Chapter 4, 2000, McGraw-Hill, NewYork, which is hereby incorporated by reference in its entirety.

In addition to conventional lithographic techniques, the apparatus ofthe present invention can be used to house next-generation lithographiessuch as extreme ultraviolet lithography, X-ray lithography (e.g., LIGA),charged-particle-beam lithography (e.g., electron-beam, ion-beam),scanning probe lithography (e.g., scanning tunneling microscopelithography, atomic force microscope lithography, scanningelectrochemical microscope lithography), soft lithography (e.g., replicamold, micro-contact printing, micro-molding in capillaries,micro-transfer molding, solvent-assisted micromolding, near-fieldconformal photolithography using an elastomeric phase-shifting mask) andthree-dimensional lithography (e.g., holographic lithography,lithography on non-planar substrates). Such techniques are described inMadou, Fundamentals of Microfabrication, Second Edition, 2002, CRC PressLLC Boca Raton, Fla., pp. 48-68, which is hereby incorporated herein byreference in its entirety.

Embodiments of the present invention can be used for protein patterningmicrolithographic techniques that address, for example, the problem ofnon-specific protein absorption competing for detection sites inimmunosensors. See, for example, Clementi et al., Structure and Motion:Membranes, Nucleic Acids, and Proteins, Adenine Press, Schenectady,N.Y., 1985, which is hereby incorporated herein by reference in itsentirety.

Devices, systems, apparatus and methods of the present invention can beused to perform dry etching techniques such as physical etching (e.g.,ion etching, sputtering, ion-beam milling), plasma etching (e.g.,radical etching), physical/chemical etching, deep reactive ion etching,vapor-phase etching without plasma, dry etching, and single-crystalreactive etching metallization (SCREAM) as disclosed in Chapter 2 ofMadou Fundamentals of Microfabrication, Second Edition, 2002, CRC PressLLC Boca Raton, Fla., which is hereby incorporated herein by referencein its entirety.

The apparatus of the present invention can be used to perform patterntransfer with additive techniques such as silicon growth, doping ofsilicon, oxidation of silicon, physical vapor diffusion (e.g., thermalevaporation, sputtering, molecular beam epitaxy, laser sputterdeposition, ablation deposition, ion plating, cluster beam technology),chemical vapor deposition, silk-screening (screen printing), sol-geldeposition, plasma spraying, spray pyrolysis, and plasma-beam depositionas disclosed in Chapter 3 of Madou Fundamentals of Microfabrication,Second Edition, 2002, CRC Press LLC Boca Raton, Fla., which is herebyincorporated herein by reference in its entirety.

Embodiments of the present invention may be particularly useful indeposition and arraying methods used in the BIOMEMS field, whichencompasses techniques for depositing organic materials for chemical andbiological sensors, often arranged in some type of array configuration.Such techniques can be used, for example, to make organic gas permeablemembranes, ion selective membranes, hydrogels, organic monolayers neededfor room-temperature gas sensors, ion selective electrodes, enzymesensors, immunosensors, and DNA and protein arrays. Specific techniquesin the BIOMEMS field that can be implemented on the apparatus of thepresent invention are spin coating, dip coating, plastic spraying,casting, type casting, glow discharge (plasma) polymerization,Langmuir-Blogett processes, ink-jetting, microspotting, and mechanicalmicrospotting, as disclosed in Madou Fundamentals of Microfabrication,Second Edition, pp. 159-167, 2002, CRC Press LLC Boca Raton, Fla., whichis hereby incorporated herein by reference in its entirety.

Embodiments of the present invention can be used for other clean roomapplications, such as packaging of small devices (e.g., integratedcircuits). Specific packaging techniques that can be performed using theapparatus of the present invention include, but are not limited to,packaging of integrated circuits, dicing, cavity sealing and bonding,multi-chip packaging, and partitioning as disclosed in MadouFundamentals of Microfabrication, Second Edition, pp. 478-508, 2002, CRCPress LLC Boca Raton, Fla., which is hereby incorporated herein byreference in its entirety.

Embodiments of the present invention can be used for application ofphotoresists in lithographic processes. For instance, the apparatus ofthe present invention can be used to apply either positive or negativephotoresist to a substrate in a clean room environment. As such, stepssuch as resist spin coating, softbake, hardbake, development,post-exposure bake, and multi-layer resists processes can be performedin the apparatus of the present invention as described in Levinson,Principles of Lithography, SPIE Press, Bellingham, Wash., 2001, Chapter3, which is hereby incorporated herein by reference in its entirety. Theapparatus of the present invention can be used for lithographicprocesses such as optical lithography, electron beam lithography, x-raylithography, deep-UV resist application, photomask fabrication, as wellas metrology methods in photolithography as described inMicrolithography, Micromachining, and Microfabrication, ed.Rai-Choudhury, SPIE Press, Bellingham, Wash., 2001, Chapters 1-6, whichis hereby incorporated herein by reference in its entirety. The systems,devices, apparatus and methods of the present invention can further beused to implement methods used to fabricate photovoltaic cells,including but not limited to phosphorous diffusion, edge isolation, ARCdeposition, front-contact printing, back-contact printing, co-firing,testing and sorting, as disclosed in Handbook of Photovoltaic Scienceand Engineering, Luque and Gegedu eds., John Wiley & Sons, West Sussex,England, 2003, pp. 271-279, which is hereby incorporated herein byreference in its entirety.

All references cited herein are incorporated herein by reference intheir entirety to the same extent as if each individual publication orpatent or patent application was specifically and individually indicatedto be incorporated by reference in its entirety. However, theincorporated by reference documents are not to be used to narrow aninterpretation of a claim element of the pending application or anypatent issuing thereon.

While the foregoing description and drawings represent embodiments ofthe present invention, it will be understood that various additions,modifications and substitutions may be made therein without departingfrom the spirit and scope of the present invention as defined in theaccompanying claims. In particular, it will be clear to those skilled inthe art that the present invention may be embodied in other specificforms, structures, arrangements, proportions, and with other elements,materials, and components, without departing from the spirit oressential characteristics thereof. The presently disclosed embodimentsare therefore to be considered in all respects as illustrative and notrestrictive, the scope of the invention being indicated by the appendedclaims, and not limited to the foregoing description.

That which is claimed:
 1. An apparatus for measuring volume of a fluid,comprising: at least one emitter configured to project a signal toward apredetermined position of a sample container; at least one receiverconfigured to receive the signal after the signal interacts with thesample container, wherein a change in the signal received by thereceiver indicates when the fluid has dropped below the predeterminedposition; and a fluid transfer device in communication with the receiverand sample container, wherein the fluid transfer device determines avolume of fluid that it has removed from the sample container when thereceiver detects that the fluid has dropped below the predeterminedposition.
 2. The apparatus of claim 1, wherein the at least one emitteris a light emitter and the signal comprises light.
 3. The apparatus ofclaim 1, wherein the fluid transfer device comprises at least a portionof a pipette.
 4. The apparatus of claim 1, wherein the sample containercomprises a cuvette.
 5. The apparatus of claim 1, further comprising aplurality of sample containers that are serially in communication withthe at least one emitter.
 6. The apparatus of claim 1, wherein thesample container is a cuvette with a prism shaped bottom.
 7. Theapparatus of claim 1, wherein the at least one emitter includes firstand second light emitters that reside on opposing sides of the samplecontainer to be able to project a respective light signal toward and inline with each other, and wherein the at least one receiver includesfirst and second infrared (IR) receivers that reside under the samplecontainer on different sides of a longitudinally extending centerline ofthe sample container, wherein the first IR receiver communicatesreceives the light signal projected from the first emitter and thesecond IR receiver receives the light signal projected from the secondemitter when the fluid has dropped below the predetermined position andthe projected signals interact with the sample container to turn 90degrees before being received by the respective IR receivers.
 8. Theapparatus of claim 1, wherein the fluid transfer device comprises apipette tip with a pipette head sealably attached to a flexible barrierthat seals off an interior of a container with the sample container heldinside the container, and wherein the pipette head receives a pipettehead adapter of a robotic arm that releasably engages a robotic arm,wherein, in operation, the robotic arm moves the pipette tip up and downand across a space inside the container while the container remainssealed.
 9. An apparatus for measuring volume of a fluid, comprising: atleast one light source for emitting light; at least one receiver forreceiving light from the light source; a cuvette residing proximate theat least one light source and the at least one receiver through which achange in a light path, dependent upon whether the cuvette containsfluid or is empty, can be detected by the at least one receiver; and afluid transfer device in communication with the receiver to determine avolume of fluid that has been removed from the cuvette when the receiverdetects that the cuvette is empty.
 10. The apparatus of claim 9, whereinthe fluid transfer device comprises at least a portion of a pipette. 11.The apparatus of claim 9, wherein the sample container is a cuvette witha prism shaped bottom.
 12. The apparatus of claim 9, wherein the atleast one light source includes first and second light sources thatreside on opposing sides of the cuvette to be able to project a lightsignal toward and in line with each other, and wherein the at least onereceiver includes first and second infrared (IR) receivers that resideunder the cuvette on different sides of a longitudinally extendingcenterline of the cuvette, wherein the first IR receiver receives lightsignal projected from the first light source and the second IR receiverreceives light signal projected from the second light source when fluidin the cuvette has dropped sufficiently low so that the projected lightsignals interact with the cuvette to turn 90 degrees before beingreceived by a respective IR receiver.
 13. The apparatus of claim 9,wherein the fluid transfer device comprises a pipette tip with a pipettehead sealably attached to a flexible barrier that seals off an interiorof a container with the cuvette held inside the container, and whereinthe pipette head receives a pipette head adapter of a robotic arm thatreleasably engages a robotic arm, wherein, in operation, the robotic armmoves the pipette tip up and down and across a space inside thecontainer while the container remains sealed.